US20110065608A1 - Devices for Detecting Renal Disorders - Google Patents

Devices for Detecting Renal Disorders Download PDF

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US20110065608A1
US20110065608A1 US12/852,312 US85231210A US2011065608A1 US 20110065608 A1 US20110065608 A1 US 20110065608A1 US 85231210 A US85231210 A US 85231210A US 2011065608 A1 US2011065608 A1 US 2011065608A1
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microglobulin
alpha
thp
kim
timp
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Samuel T. Labrie
James P. Mapes
Ralph L. McDade
Dominic P. Eisinger
Karri L. Ballard
Michael D. Spain
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Myriad RBM Inc
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Rules Based Medicine Inc
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Definitions

  • the invention encompasses devices for diagnosing, monitoring, or determining a renal disorder in a mammal.
  • the present invention provides methods and devices for diagnosing, monitoring, or determining renal disorders in a mammal using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.
  • the urinary system in particular the kidneys, perform several critical functions such as maintaining electrolyte balance and eliminating toxins from the bloodstream.
  • the pair of kidneys together process roughly 20% of the total cardiac output, amounting to about 1 L/min in a 70-kg adult male. Because compounds in circulation are concentrated in the kidney up to 1000-fold relative to the plasma concentration, the kidney is especially vulnerable to injury due to exposure to toxic compounds.
  • Renal disorders and disease are serious conditions that generally affect the function of the kidney.
  • the disorders discussed herein may arise from a variety of causes, including intrinsic disease processes, such as inflammation and necrosis of the kidney.
  • renal disorders may also arise from secondary sources including drugs that are toxic to the kidneys and alternative disease states that cause secondary adverse effects on the kidney, such as diabetes and hypertension.
  • Prevention of renal disorders is largely dependent on early diagnosis of the condition.
  • Existing diagnostic tests such as BUN and serum creatine tests typically detect only advanced stages of kidney damage.
  • Other diagnostic tests such as kidney tissue biopsies or CAT scans have the advantage of enhanced sensitivity to earlier stages of kidney damage, but these tests are also generally costly, slow, and/or invasive.
  • the early detection of kidney damage would help medical practitioners to diagnose and treat kidney damage more quickly and effectively.
  • the present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder in a mammal.
  • the present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.
  • the present invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
  • the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C,
  • the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising: (a) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol; (b) three or more capture agents comprising an anti
  • the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal
  • the kit includes: (a) an assay device having a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; and (b) a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
  • the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having (i) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; (ii) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (iii) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (iv) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies;
  • the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • the invention encompasses a platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • FIG. 1 depicts four graphs comparing (A) the concentrations of alpha-1 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (B) the concentrations of beta-2 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (C) the concentrations of NGAL in the urine of normal controls, kidney cancer patients, and patients with other cancer types; and (D) the concentrations of THP in the urine of normal controls, kidney cancer patients, and patients with other cancer types.
  • FIG. 2 shows the four different disease groups from which samples were analyzed, and a plot of two different estimations on eGFR outlining the distribution within each group.
  • FIG. 3 is a number of scatter plots of results on selected proteins in urine and plasma. The various groups are indicated as follows—control: blue, AA: red, DN: green, GN: yellow, OU: orange.
  • A1M in plasma (B) cystatin C in plasma, (C) B2M in urine, (D) cystatin C in urine.
  • FIG. 4 depicts the multivariate analysis of the disease groups and their respective matched controls using plasma results. Relative importance shown using the random forest model.
  • FIG. 5 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish disease samples vs. normal samples.
  • FIG. 6 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish disease (AA+GN+ON+DN) samples vs. normal samples from plasma (A) and urine (B and C).
  • FIG. 7 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. normal samples. Abbreviations as in FIG. 4 .
  • FIG. 8 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. normal samples from plasma (A) and urine (B and C).
  • FIG. 9 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. diabetic nephropathy samples. Abbreviations as in FIG. 4 .
  • FIG. 10 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. diabetic nephropathy samples from plasma (A) and urine (B and C).
  • FIG. 11 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish glomerulonephritis samples vs. analgesic abuse samples. Abbreviations as in FIG. 4 .
  • FIG. 12 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish glomerulonephritis samples vs. analgesic abuse samples from plasma (A) and urine (B and C).
  • FIG. 13 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish obstructive uropathy samples vs. analgesic abuse samples. Abbreviations as in FIG. 4 .
  • FIG. 14 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish obstructive uropathy samples vs. analgesic abuse samples from plasma (A) and urine (B and C).
  • renal disorder includes, but is not limited to glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, diabetic nephropathy, analgesic nephropathy, and acute tubular necrosis.
  • glomerulonephritis refers to a disorder characterized by inflammation of the glomeruli.
  • the term may encompass chronic glomerulonephritis, acute glomerulonephritis, primary glomerulonephritis, or secondary glomerulonephritis.
  • diabetes nephropathy refers to a disorder characterized by angiopathy of capillaries in the kidney glomeruli.
  • the term encompasses Kimmelstiel-Wilson syndrome, or nodular diabetic glomerulosclerosis and intercapillary glomerulonephritis.
  • the present invention encompasses biomarkers that may be used to detect a disorder associated with diabetic nephropathy.
  • a disorder associated with diabetic nephropathy refers to a disorder that stems from angiopathy of capillaries in the kidney glomeruli.
  • associated disorders may include nephritic syndrome, chronic kidney failure, and end-stage kidney disease.
  • the devices of the present invention may also be used to detect secondary kidney damage or toxicity caused by exposure to a toxic compound including but not limited to therapeutic drugs, recreational drugs, medical imaging contrast agents, and toxins.
  • therapeutic drugs may include an analgesic (e.g. aspirin, acetaminophen, ibuprofen, naproxen sodium), an antibiotic (e.g.
  • a chemotherapy agent e.g.
  • Cisplatin (Platinol®), Carboplatin (Paraplatin®), Cytarabine (Cytosar-U®), Gemtuzumab ozogamicin (Mylotarg®), Gemcitabine (Gemzar®), Melphalan (Alkeran®), Ifosfamide (Ifex®), Methotrexate (Rheumatrex®), Interleukin-2 (Proleukin®), Oxaliplatin (Eloxatin®), Streptozocin (Zanosar®), Pemetrexed (Alimta®), Plicamycin (Mithracin®), and Trimetrexate (Neutrexin®).
  • renal disorder may include kidney damage due to kidney stones, ischemia, liver transplantation, heart transplantation, lung transplantation, or hypovolemia.
  • the devices of the current invention may be used to detect renal disorders including kidney damage cause by other disease states including but not limited to diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to any infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate disease.
  • the devices and systems of the current invention may be used to detect renal disorders including acute kidney transplant rejection or chronic allograft nephropathy.
  • the devices of the invention may be used to distinguish between an acute rejection reaction and a chronic allograft nephropathy.
  • the devices of the present invention may be used to distinguish between a successful transplant and rejection.
  • rejection refers to a recipient response to a foreign antigen derived from the transplanted kidney.
  • acute rejection refers to an immune related response to the foreign kidney. The response is primarily T-cell driven and originates from an HLC mismatch between the donor and recipient.
  • chronic allograft nephropathy refers to a chronic inflammatory and immune response mediated reaction to a foreign kidney. Chronic allograft nephropathy may result in damage to the kidney manifested by diffuse interstitial fibrosis glomerular changes, typically membranous and sclerotic in nature, as well as intimal fibrosis of the blood vessels with tubular atrophy and loss of tubular structures.
  • the present invention encompasses devices comprising biomarkers that may be used to detect a renal disorder associated with kidney transplant rejection.
  • a disorder associated with kidney transplant rejection refers to a disorder that stems from a host response to a foreign antigen derived from the transplated kidney.
  • associated disorders may include chronic kidney failure and end-stage kidney disease.
  • the devices of the present invention may also be utilized to detect a renal disorder including obstructive uropathy or an associated disorder in a mammal that includes determining the presence or concentration of a combination of three or more sample analytes in a test sample containing the bodily fluid of the mammal.
  • obstructive uropathy refers to a structural or functional hindrance of normal urine flow. The term may encompass chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, or acute bilateral obstructive uropathy.
  • the present invention encompasses biomarkers that may be used to detect a disorder associated with obstructive uropathy.
  • a disorder associated with obstructive uropathy refers to a disorder that stems from a structural or functional hindrance of normal urine flow.
  • associated disorders may include hydronephrosis and obstructive nephropathy.
  • the measured concentrations of the combination of sample analytes is compared to the entries of a dataset in which each entry contains the minimum diagnostic concentrations of a combination of three of more analytes reflective of obstructive uropathy or an associated disorder.
  • Other embodiments provide computer-readable media encoded with applications containing executable modules, systems that include databases and processing devices containing executable modules configured to diagnose, monitor, or determine a renal disorder in a mammal.
  • Still other embodiments provide antibody-based devices for diagnosing, monitoring, or determining obstructive uropathy or an associated disorder in a mammal.
  • the biomarkers included in a multiplexed panel of the invention are analytes known in the art that may be detected in the urine, serum, plasma and other bodily fluids of mammals.
  • the analytes of the multiplexed panel may be readily extracted from the mammal in a test sample of bodily fluid.
  • the concentrations of the analytes within the test sample may be measured using known analytical techniques such as a multiplexed antibody-based immunological assay.
  • the combination of concentrations of the analytes in the test sample may be compared to empirically determined combinations of minimum diagnostic concentrations and combinations of diagnostic concentration ranges associated with healthy kidney function to determine whether a renal disorder is indicated in the mammal.
  • analytes used as biomarkers in the multiplexed assay methods of diagnosing, monitoring, or determining a renal disorder using measurements of the analytes, systems and applications used to analyze the multiplexed assay measurements, and antibody-based devices used to measure the analytes are described in detail below.
  • One embodiment of the invention measures the concentrations of three or more, six or more, ten or more, and preferably sixteen, biomarker analytes within a test sample taken from a mammal and compares the measured analyte concentrations to minimum diagnostic concentrations to diagnose, monitor, or determine obstructive uropathy or an associated renal disorder in a mammal.
  • the biomarker analytes are known in the art to occur in the urine, plasma, serum and other bodily fluids of mammals.
  • the biomarker analytes are proteins that have known and documented associations with early renal damage in humans.
  • the biomarker analytes include but are not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • a description of each biomarker analyte is given below.
  • the biomarker analytes include alpha-1-microglobulin, beta-2-microglobulin, cystatin-C, KIM-1, THP, and TIMP-1.
  • Alpha-1 microglobulin (A1M, Swiss-Prot Accession Number P02760) is a 26 kDa glycoprotein synthesized by the liver and reabsorbed in the proximal tubules. Elevated levels of A1M in human urine are indicative of glomerulotubular dysfunction. A1M is a member of the lipocalin super family and is found in all tissues. Alpha-1-microglobulin exists in blood in both a free form and complexed with immunoglobulin A (IgA) and heme. Half of plasma A1M exists in a free form, and the remainder exists in complexes with other molecules including prothrombin, albumin, immunoglobulin A and heme.
  • IgA immunoglobulin A
  • Half of plasma A1M exists in a free form, and the remainder exists in complexes with other molecules including prothrombin, albumin, immunoglobulin A and heme.
  • A1M A1M in human urine
  • proximal tubular cells Nearly all of the free A1M in human urine is reabsorbed by the megalin receptor in proximal tubular cells, where it is then catabolized. Small amounts of A1M are excreted in the urine of healthy humans. Increased A1M concentrations in human urine may be an early indicator of renal damage, primarily in the proximal tubule.
  • Beta-2 Microglobulin (b) Beta-2 Microglobulin (B2M)
  • Beta-2 microglobulin (B2M, Swiss-Prot Accession Number P61769) is a protein found on the surfaces of all nucleated cells and is shed into the blood, particularly by tumor cells and lymphocytes. Due to its small size, B2M passes through the glomerular membrane, but normally less than 1% is excreted due to reabsorption of B2M in the proximal tubules of the kidney. Therefore, high plasma levels of B2M occur as a result of renal failure, inflammation, and neoplasms, especially those associated with B-lymphocytes.
  • Calbindin (Calbindin D-28K, Swiss-Prot Accession Number P05937) is a Ca-binding protein belonging to the troponin C superfamily. It is expressed in the kidney, pancreatic islets, and brain. Calbindin is found predominantly in subpopulations of central and peripheral nervous system neurons, in certain epithelial cells involved in Ca2+ transport such as distal tubular cells and cortical collecting tubules of the kidney, and in enteric neuroendocrine cells.
  • Clusterin (Swiss-Prot Accession Number P10909) is a highly conserved protein that has been identified independently by many different laboratories and named SGP2, S35-S45, apolipoprotein J, SP-40, 40, ADHC-9, gp80, GPIII, and testosterone-repressed prostate message (TRPM-2).
  • SGP2 S35-S45
  • apolipoprotein J SP-40
  • 40 ADHC-9
  • gp80 gp80
  • GPIII testosterone-repressed prostate message
  • TRPM-2 testosterone-repressed prostate message
  • clusterin protein has also been implicated in physiological processes that do not involve apoptosis, including the control of complement-mediated cell lysis, transport of beta-amyloid precursor protein, shuttling of aberrant beta-amyloid across the blood-brain barrier, lipid scavenging, membrane remodeling, cell aggregation, and protection from immune detection and tumor necrosis factor induced cell death.
  • CTGF Connective Tissue Growth Factor
  • CTGF Connective tissue growth factor
  • P29279 Connective tissue growth factor
  • Creatinine is a metabolite of creatine phosphate in muscle tissue, and is typically produced at a relatively constant rate by the body. Creatinine is chiefly filtered out of the blood by the kidneys, though a small amount is actively secreted by the kidneys into the urine. Creatinine levels in blood and urine may be used to estimate the creatinine clearance, which is representative of the overall glomerular filtration rate (GFR), a standard measure of renal function. Variations in creatinine concentrations in the blood and urine, as well as variations in the ratio of urea to creatinine concentration in the blood, are common diagnostic measurements used to assess renal function.
  • GFR overall glomerular filtration rate
  • Cystatin C (Cyst C, Swiss-Prot Accession Number P01034) is a 13 kDa protein that is a potent inhibitor of the C1 family of cysteine proteases. It is the most abundant extracellular inhibitor of cysteine proteases in testis, epididymis, prostate, seminal vesicles and many other tissues. Cystatin C, which is normally expressed in vascular wall smooth muscle cells, is severely reduced in both atherosclerotic and aneurismal aortic lesions.
  • Epidermal growth factor (EGF, Swiss-Prot Accession Number P07522) is a small protein that functions as a potent mitogen. EGF promotes cell growth and differentiation, is essential in embryogenesis, and is important in wound healing. It is produced by many normal cell types and is made in large amounts by certain types of tumors.
  • Glutathione S-transferase alpha (GST-alpha, Swiss-Prot Accession Number P08263) belongs to a family of enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of compounds, including carcinogens, therapeutic drugs, and products of oxidative stress. These enzymes play a key role in the detoxification of such substances.
  • Glutathione S-transferase mu functions in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione.
  • the genes encoding the mu class of enzymes are organized in a gene cluster on chromosome 1 p13.3 and are known to be highly polymorphic. Genetic variations in GST-mu can change a mammal's susceptibility to carcinogens and toxins as well as affect the toxicity and efficacy of certain drugs. Null mutations of this class mu gene have been linked with an increase in a number of cancers.
  • Kidney injury molecule-1 (KIM-1, Swiss-Prot Accession Number Q96D42) is an immunoglobulin superfamily cell-surface protein highly upregulated on the surface of injured kidney epithelial cells. It is also known as TIM-1 (T-cell immunoglobulin mucin domain-1), as it is expressed at low levels by subpopulations of activated T-cells and hepatitis A virus cellular receptor-1 (HAVCR-1). KIM-1 is increased in expression more than any other protein in the injured kidney and is localized predominantly to the apical membrane of the surviving proximal epithelial cells.
  • TIM-1 T-cell immunoglobulin mucin domain-1
  • HAVCR-1 hepatitis A virus cellular receptor-1
  • Albumin is the most abundant plasma protein in humans and other mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. Healthy, normal kidneys typically filter out albumin from the urine. The presence of albumin in the urine may indicate damage to the kidneys. Albumin in the urine may also occur in patients with long-standing diabetes, especially type 1 diabetes. The amount of albumin eliminated in the urine has been used to differentially diagnose various renal disorders. For example, nephrotic syndrome usually results in the excretion of about 3.0 to 3.5 grams of albumin in human urine every 24 hours. Microalbuminuria, in which less than 300 mg of albumin is eliminated in the urine every 24 hours, may indicate the early stages of diabetic nephropathy.
  • Neutrophil gelatinase-associated lipocalin (NGAL, Swiss-Prot Accession Number P80188) forms a disulfide bond-linked heterodimer with MMP-9. It mediates an innate immune response to bacterial infection by sequestrating iron. Lipocalins interact with many different molecules such as cell surface receptors and proteases, and play a role in a variety of processes such as the progression of cancer and allergic reactions.
  • Osteopontin (OPN, Swiss-Prot Accession Number P10451) is a cytokine involved in enhancing production of interferon-gamma and IL-12, and inhibiting the production of IL-10.
  • OPN is essential in the pathway that leads to type I immunity.
  • OPN appears to form an integral part of the mineralized matrix.
  • OPN is synthesized within the kidney and has been detected in human urine at levels that may effectively inhibit calcium oxalate crystallization. Decreased concentrations of OPN have been documented in urine from patients with renal stone disease compared with normal individuals.
  • Tamm-Horsfall protein (THP, Swiss-Prot Accession Number P07911), also known as uromodulin, is the most abundant protein present in the urine of healthy subjects and has been shown to decrease in individuals with kidney stones.
  • THP is secreted by the thick ascending limb of the loop of Henley.
  • THP is a monomeric glycoprotein of ⁇ 85 kDa with ⁇ 30% carbohydrate moiety that is heavily glycosylated.
  • THP may act as a constitutive inhibitor of calcium crystallization in renal fluids.
  • Tissue Inhibitor of Metalloproteinase-1 (p) Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)
  • Tissue inhibitor of metalloproteinase-1 (TIMP-1, Swiss-Prot Accession Number P01033) is a major regulator of extracellular matrix synthesis and degradation.
  • a certain balance of MMPs and TIMPs is essential for tumor growth and health. Fibrosis results from an imbalance of fibrogenesis and fibrolysis, highlighting the importance of the role of the inhibition of matrix degradation role in renal disease.
  • Trefoil factor 3 (TFF3, Swiss-Prot Accession Number Q07654), also known as intestinal trefoil factor, belongs to a small family of mucin-associated peptides that include TFF1, TFF2, and TFF3.
  • TFF3 exists in a 60-amino acid monomeric form and a 118-amino acid dimeric form. Under normal conditions TFF3 is expressed by goblet cells of the intestine and the colon. TFF3 expression has also been observed in the human respiratory tract, in human goblet cells and in the human salivary gland. In addition, TFF3 has been detected in the human hypothalamus.
  • VEGF Vascular Endothelial Growth Factor
  • VEGF Vascular endothelial growth factor
  • VEGF A Vascular Endothelial Growth Factor A
  • Vascular endothelial growth factor A (VEGF A, Swiss-Prot Accession Number Q00731) is a growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. It induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces permeabilization of blood vessles. It is important in the pathophysiology of neuronal and other tumors, likely functioning as a potent promoter of angiogenesis. Due to its influences on vascular permeability, VEGF A may be involved in altering blood-brain-barrier functions under normal and pathological conditions. The production and secretion of VEGF by mammalian retinal pigment epithelial cells may be important in the pathogenesis of ocular neovascularization.
  • B-lymphocyte chemoattractant (BLC, Swiss-Prot Accession Number 043927) is also referred to as C-X-C motif chemokine 13, Small-inducible cytokine B13, B lymphocyte chemoattractant, CXC chemokine BLC, and B cell-attracting chemokine 1.
  • BLC functions as a potent chemoattractant for B lymphocytes, but not T lymphocytes, monocytes, or neutrophils.
  • Its specific receptor BLR1 is a G protein-coupled receptor originally isolated from Burkitt's lymphoma cells. Among cells of the hematopoietic lineages, the expression of BRL1, now designated CXCR5, is restricted to B lymphocytes and a subpopulation of T helper memory cells.
  • CD40 Cluster of Differentiation Surface Receptors 40
  • TNFRSF5 Tumor necrosis factor receptor superfamily member 5.
  • CD40 is a member of the tumor necrosis factor-receptor superfamily of proteins. CD40 has been found to be essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.
  • IGF BP2 Insulin-Like Growth Factor Binding Protein 2
  • Insulin-like Growth Factor Binding Protein 2 (IGF BP2, Swiss Prot Accession Number P18065) functions to prolong the half-life of the insulin growth factors and have been shown to either inhibit or stimulate the growth promoting effects of the insulin growth factors on cell culture. Specifically, during development, insulin-like growth factor binding protein-2 is expressed in a number of tissues with the highest expression level found in the central nervous system. IGFBP-2 exhibits a 2-10 fold higher affinity for IGF II than for IGF I.
  • Matrix Metalloproteinase-3 (MMP3, Swiss Prot Accession Number P08254) is also known as stromelysin-1 and Transin-1.
  • MMP3 is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases.
  • MMP3 encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. The enzyme is thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation.
  • MMP3 is part of a cluster of MMP genes which localize to chromosome 11q22.3.
  • Peptide YY (PYY, Swiss-Prot Accession Number P10082) is also known as peptide tyrosine tyrosine and pancreatic peptide YY 3-36 .
  • Peptide YY exerts its action through neuropeptide Y receptors, inhibits gastric motility and increases water and electrolyte absorption in the colon.
  • PYY may also suppress pancreatic secretion. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite.
  • PYYY works by slowing the gastric emptying; hence, it increases efficiency of digestion and nutrient absorption after meal. Research has also indicated that PYY may be useful in removing aluminum accumulated in the brain.
  • SCF Stem Cell Factor
  • Stem Cell Factor (SCF, UniProtKB/TrEMBL Q13528) is also known as kit-ligand, KL, and steel factor. SCF functions SCF plays an important role in the hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and decreased pigmentation.
  • HSCs hematopoietic stem cells
  • melanocytes cells that produce melanin and control pigmentation.
  • melanogenisis melanoblasts migrate from the neural crest to their appropriate locations in the epidermis. Melanoblasts express the Kit receptor, and it is believed that SCF guides these cells to their terminal locations. SCF also regulates survival and proliferation of fully differentiated melanocytes in adults.
  • c-Kit is expressed in primordial germ cells, spermatogonia, and in primordial oocytes. It is also expressed in the primordial germ cells of females. SCF is expressed along the pathways that the germ cells use to reach their terminal destination in the body. It is also expressed in the final destinations for these cells. Like for melanoblasts, this helps guide the cells to their appropriate locations in the body
  • TNF RII Tumor Necrosis Factor Receptor Type II
  • Tumor Necrosis Factor Receptor Type II (TNF RII, Swiss-Prot Accession Number P20333) is also known as p75, p80 TNF alpha receptor, and TNFRSF1B.
  • TNF RII is a protein that in humans is encoded by the TNFRSF1B gene.
  • the protein encoded by this gene is a member of the Tumor necrosis factor receptor superfamily, which also contains TNFRSF1A.
  • the protein encoded by this gene is a member of the TNF-receptor superfamily.
  • This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity.
  • the function of IAPs in TNF-receptor signaling is unknown; however, c-IAP1 is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2, which mediates anti-apoptotic signals.
  • Knockout studies in mice also suggest a role of this protein in protecting neurons from apoptosis by stimulating antioxidative pathways.
  • AXL (Swiss-Prot Accession Number P30530) is also known as UFO, ARK, and tyrosine-protein kinase receptor UFO.
  • the protein encoded by AXL is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the AXL protein represents a unique structure of the extracellular region that juxtaposes IgL and FNIII repeats.
  • AXL transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin K-dependent protein growth-arrest-specific gene 6. It is involved in the stimulation of cell proliferation. This receptor can also mediate cell aggregation by homophilic binding.
  • AXL is a chronic myelogenous leukemia-associated oncogene and also associated with colon cancer and melanoma.
  • Eotaxin 3 (Swiss-Prot Accession Number P51671) is also known as C-C motif chemokine 11 (CCL11), small inducible cytokine A11, and eosinophil chemotactic protein.
  • Eotaxin 3 is a small cytokine belonging to the CC chemokine family that is also called Eotaxin-3, Macrophage inflammatory protein 4-alpha (MIP-4-alpha), Thymic stroma chemokine-1 (TSC-1), and IMAC.
  • CCL26 cytokine interleukin 4.[1][2] CCL26 is chemotactic for eosinophils and basophils and elicits its effects by binding to the cell surface chemokine receptor CCR3.
  • Fatty Acid Binding Protein (FABP, Swiss-Prot Accession Number Q01469) is also known as epidermal-type fatty acid binding protein, and fatty-acid binding protein 5. This gene encodes the fatty acid binding protein found in epidermal cells, and was first identified as being upregulated in psoriasis tissue. Fatty acid binding proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism.
  • Basic Fibroblast Growth Factor (FGF basic, Swiss-Prot Accession NumberP09038) is also known as heparin-binding growth factor.
  • basic fibroblast growth factor In normal tissue, basic fibroblast growth factor is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It stays membrane-bound as long as there is no signal peptide. It has been hypothesized that, during both wound healing of normal tissues and tumor development, the action of heparan sulfate-degrading enzymes activates FGF basic, thus mediating the formation of new blood vessels.
  • FGF basic is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by bone morphogenetic proteins. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems.
  • Myoglobin (Swiss-Prot Accession Number P02144) is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may cause acute renal failure. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest pain.
  • Resistin (RETN, UniProtKB/TrEMBL Q76B53) is theorized to participate in the inflammatory response. Resistin has also been shown to increase transcriptional events leading to an increased expression of several pro-inflammatory cytokines including (but not limited to) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor- ⁇ (TNF- ⁇ ) in an NF-KB-mediated fashion. It has also been demonstrated that resistin upregulates intracellular adhesion molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and CCL2, all of which are occupied in chemotactic pathways involved in leukocyte recruitment to sites of infection.
  • IAM1 intracellular adhesion molecule-1
  • VCAM1 vascular cell-adhesion molecule-1
  • CCL2 CCL2
  • Resistin itself can be upregulated by interleukins and also by microbial antigens such as lipopolysaccharide, which are recognized by leukocytes.
  • microbial antigens such as lipopolysaccharide, which are recognized by leukocytes.
  • TRAIL R3 (Swiss-Prot Accession Number P83626 (mouse)) is also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 3, and tumor necrosis factor receptor mouse homolog.
  • TRAIL R3 is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors.
  • Endothelin 1 (ET1, UniProtKB/TrEMBL Q6FH53) is also known as EDN1 and EDN1 protein. Endothelin 1 is a protein that constricts blood vessels and raises blood pressure. It is normally kept in balance by other mechanisms, but when over-expressed, it contributes to high blood pressure (hypertension) and heart disease. Endothelin 1 peptides and receptors are implicated in the pathogenesis of a number of disease states, including cancer and heart disease.
  • Neuronal Cell Adhesion Molecule (NrCAM, UniProtKB/TrEMBL Q14CA1) encodes a neuronal cell adhesion molecule with multiple immunoglobulin-like C2-type domains and fibronectin type-III domains. This ankyrin-binding protein is involved in neuron-neuron adhesion and promotes directional signaling during axonal cone growth. This gene is also expressed in non-neural tissues and may play a general role in cell-cell communication via signaling from its intracellular domain to the actin cytoskeleton during directional cell migration. Allelic variants of this gene have been associated with autism and addiction vulnerability.
  • Tenascin C (TN-C, UniProt/TrEMBL Q99857) has anti-adhesive properties, causing cells in tissue culture to become rounded after it is added to the medium.
  • One mechanism to explain this may come from its ability to bind to the extracellular matrix glycoprotein fibronectin and block fibronectin's interactions with specific syndecans.
  • the expression of tenascin-C in the stroma of certain tumors is associated with a poor prognosis.
  • VCAM1 Vascular Cell Adhesion Molecule 1
  • Vascular Cell Adhesion Molecule 1 (VCAM1, Swiss-Prot Accession Number P19320) is also known as vascular cell adhesion protein 1. VCAM1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis.
  • VCAM-1 Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNF- ⁇ ) and Interleukin-1 (IL-1)) and through stabilization of Messenger RNA (mRNA) (e.g., Interleukin-4 (IL-4)).
  • mRNA Messenger RNA
  • IL-4 Interleukin-4
  • the promoter region of the VCAM-1 gene contains functional tandem NF- ⁇ B (nuclear factor-kappa B) sites.
  • the sustained expression of VCAM-1 lasts over 24 hours.
  • VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or ⁇ 4 ⁇ 1) of the ⁇ 1 subfamily of integrins, and for integrin ⁇ 4 ⁇ 7.
  • VCAM-1 expression has also been observed in other cell types (e.g., smooth muscle cells). It has also been shown to interact with EZR and Moesin. Certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites.
  • Cortisol (Swiss-Prot Accession Number P08185) is also known as corticosteroid-binding globulin, transcortin, and Serpin A6.
  • Cortisol is a steroid hormone or glucocorticoid produced by the adrenal gland. It is released in response to stress, and to a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis, suppress the immune system, and aid in fat, protein and carbohydrate metabolism. It also decreases bone formation. In addition, cortisol can weaken the activity of the immune system.
  • Cortisol prevents proliferation of T-cells by rendering the interleukin-2 producer T-cells unresponsive to interleukin-1 (IL-1), and unable to produce the T-cell growth factor.
  • Cortisol also has a negative feedback effect on interleukin-1.
  • IL-1 must be especially useful in combating some diseases; however, endotoxin bacteria have gained an advantage by forcing the hypothalamus to increase cortisol levels via forcing secretion of CRH hormone, thus antagonizing IL-1 in this case.
  • the suppressor cells are not affected by GRMF, so that the effective set point for the immune cells may be even higher than the set point for physiological processes. It reflects leukocyte redistribution to lymph nodes, bone marrow, and skin.
  • the device for diagnosing, monitoring, or determining a renal disorder involves determining the presence or concentrations of a combination of sample analytes in a test sample.
  • the combinations of sample analytes are any group of three or more analytes selected from the biomarker analytes, including but not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol.
  • the combination of analytes may be selected to provide a
  • the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes.
  • the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, or all sixteen of the sixteen biomarker analytes.
  • the combination of sample analytes may comprise a combination listed in Table A.
  • the combination of sample analytes may include creatinine, KIM-1, and THP. In another exemplary embodiment, the combination of sample analytes may include microalbumin, creatinine, and KIM-1. In yet another exemplary embodiment, the combination of sample analytes may include creatinine, THP, and A1M. In still another exemplary embodiment, the combination of sample analytes may include microalbumin, TIMP-1, and osteopontin.
  • the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of obstructive uropathy.
  • the combination of sample analytes may include any three of the biomarker analytes previously discussed.
  • the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy include three or more biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin.
  • the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy includes six biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin.
  • the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of glomerulonephritis.
  • the combination of sample analytes may include any three of the biomarker analytes previously discussed.
  • the devices and systems to diagnose, monitor or determine the presence of glomerulonephritis include three or more biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
  • the devices and systems to diagnose, monitor or determine the presence of glomerulonephropathy includes six biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
  • the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of kidney damage or toxicity.
  • the combination of sample analytes may include any three of the biomarker analytes previously discussed.
  • the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include three or more biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
  • the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include six biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
  • the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of diabetic nephropathy.
  • the combination of sample analytes may include any three of the biomarker analytes previously discussed.
  • the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include three or more biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
  • the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include six biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
  • the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes discussed previously to diagnose kidney transplant rejection or other associated disease as discussed previously.
  • the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, any seventeen, any eighteen, or any nineteen biomarker analytes.
  • the combination of sample analytes may comprise a combination listed in Table B.
  • test sample is an amount of bodily fluid taken from a mammal.
  • bodily fluids include urine, blood, plasma, serum, saliva, semen, perspiration, tears, mucus, and tissue lysates.
  • the bodily fluid contained in the test sample is urine, plasma, or serum.
  • a mammal as defined herein, is any organism that is a member of the class Mammalia.
  • mammals appropriate for the various embodiments may include humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen.
  • the mammal is a human.
  • the bodily fluids of the test sample may be taken from the mammal using any known device or method so long as the analytes to be measured by the multiplexed assay are not rendered undetectable by the multiplexed assay.
  • devices or methods suitable for taking bodily fluid from a mammal include urine sample cups, urethral catheters, swabs, hypodermic needles, thin needle biopsies, hollow needle biopsies, punch biopsies, metabolic cages, and aspiration.
  • the test sample may be diluted to reduce the concentration of the sample analytes prior to analysis.
  • the degree of dilution may depend on a variety of factors including but not limited to the type of multiplexed assay used to measure the analytes, the reagents utilized in the multiplexed assay, and the type of bodily fluid contained in the test sample.
  • the test sample is diluted by adding a volume of diluent ranging from about 1 ⁇ 2 of the original test sample volume to about 50,000 times the original test sample volume.
  • test sample is human urine and the multiplexed assay is an antibody-based capture-sandwich assay
  • the test sample is diluted by adding a volume of diluent that is about 100 times the original test sample volume prior to analysis.
  • test sample is human serum and the multiplexed assay is an antibody-based capture-sandwich assay
  • the test sample is diluted by adding a volume of diluent that is about 5 times the original test sample volume prior to analysis.
  • test sample is human plasma and the multiplexed assay is an antibody-based capture-sandwich assay
  • the test sample is diluted by adding a volume of diluent that is about 2,000 times the original test sample volume prior to analysis.
  • the diluent may be any fluid that does not interfere with the function of the multiplexed assay used to measure the concentration of the analytes in the test sample.
  • suitable diluents include deionized water, distilled water, saline solution, Ringer's solution, phosphate buffered saline solution, TRIS-buffered saline solution, standard saline citrate, and HEPES-buffered saline.
  • the concentration of a combination of sample analytes is measured using a multiplexed assay device capable of measuring up to 189 of the biomarker analytes.
  • a multiplexed assay device is an assay capable of simultaneously determining the concentration of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more of the biomarker analytes using a single device and/or method.
  • any known method of measuring the concentration of the biomarker analytes may be used for the multiplexed assay device.
  • measurement methods suitable for the multiplexed assay device include electrophoresis, mass spectrometry, protein microarrays, surface plasmon resonance, and immunoassays including, but not limited to western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA) methods, vibrational detection using MicroElectroMagnetic Devices (MEMS), and particle-based capture-sandwich immunoassays.
  • the concentrations of the analytes in the test sample are measured using a multiplexed immunoassay device that utilizes capture antibodies marked with indicators to determine the concentration of the sample analytes.
  • the multiplexed immunoassay device includes three or more capture antibodies.
  • Capture antibodies are antibodies in which the antigenic determinant is one of the Biomarker Analytes known in the art to have a documented association with early renal damage in humans.
  • the biomarker analytes include, but are note limited to alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • Each of the at least three capture antibodies has a unique antigenic determinant that is one of the biomarker analytes. When contacted with the test sample, the capture antibodies form antigen-antibody complexes in which the analytes serve as antigens.
  • antibody encompasses a monoclonal ab, an antibody fragment, a chimeric antibody, and a single-chain antibody.
  • the capture antibodies may be attached to a platform or other substrate having a contact surface in order to immobilize any analytes captured by the capture antibodies.
  • the platform generally incorporates a porous material for immobilizing the analytes.
  • suitable substrates include paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, or plastic strips, beads, or surfaces, such as the inner surface of the well of a microtitration tray.
  • Suitable beads may include polystyrene or latex microspheres.
  • an indicator is attached to each of the three or more capture antibodies.
  • the indicator as defined herein, is any compound that registers a measurable change to indicate the presence of one of the sample analytes when bound to one of the capture antibodies.
  • Non-limiting examples of indicators include visual indicators and electrochemical indicators.
  • Visual indicators are compounds that register a change by reflecting a limited subset of the wavelengths of light illuminating the indicator, by fluorescing light after being illuminated, or by emitting light via chemiluminescence.
  • the change registered by visual indicators may be in the visible light spectrum, in the infrared spectrum, or in the ultraviolet spectrum.
  • Non-limiting examples of visual indicators suitable for the multiplexed immunoassay device include nanoparticulate gold, organic particles such as polyurethane or latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, quantum dots, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored or chemiluminescent product.
  • Electrochemical indicators are compounds that register a change by altering an electrical property.
  • the changes registered by electrochemical indicators may be an alteration in conductivity, resistance, capacitance, current conducted in response to an applied voltage, or voltage required to achieve a desired current.
  • Non-limiting examples of electrochemical indicators include redox species such as ascorbate (vitamin C), vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron and mercury.
  • test sample containing a combination of three or more sample analytes is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as the antigens.
  • concentrations of the three or more analytes are determined by measuring the change registered by the indicators attached to the capture antibodies.
  • the indicators are polyurethane or latex microspheres loaded with dye compounds and phycoerythrin.
  • the multiplexed immunoassay device has a sandwich assay format.
  • the multiplexed sandwich immunoassay device includes three or more capture antibodies as previously described. However, in this embodiment, each of the capture antibodies is attached to a capture agent that includes an antigenic moiety. The antigenic moiety serves as the antigenic determinant of a detection antibody, also included in the multiplexed immunoassay device of this embodiment. In addition, an indicator is attached to the detection antibody.
  • the test sample is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as antigens.
  • the detection antibodies are then contacted with the test sample and allowed to form antigen-antibody complexes in which the capture agent serves as the antigen for the detection antibody. After removing any uncomplexed detection antibodies the concentration of the analytes are determined by measuring the changes registered by the indicators attached to the detection antibodies.
  • the concentrations of each of the sample analytes may be determined using any approach known in the art.
  • a single indicator compound is attached to each of the three or more antibodies.
  • each of the capture antibodies having one of the sample analytes as an antigenic determinant is physically separated into a distinct region so that the concentration of each of the sample analytes may be determined by measuring the changes registered by the indicators in each physically separate region corresponding to each of the sample analytes.
  • each antibody having one of the sample analytes as an antigenic determinant is marked with a unique indicator.
  • a unique indicator is attached to each antibody having a single sample analyte as its antigenic determinant.
  • all antibodies may occupy the same physical space. The concentration of each sample analyte is determined by measuring the change registered by the unique indicator attached to the antibody having the sample analyte as an antigenic determinant.
  • the multiplexed immunoassay device is a microsphere-based capture-sandwich immunoassay device.
  • the device includes a mixture of three or more capture-antibody microspheres, in which each capture-antibody microsphere corresponds to one of the biomarker analytes.
  • Each capture-antibody microsphere includes a plurality of capture antibodies attached to the outer surface of the microsphere.
  • the antigenic determinant of all of the capture antibodies attached to one microsphere is the same biomarker analyte.
  • the microsphere is a small polystyrene or latex sphere that is loaded with an indicator that is a dye compound.
  • the microsphere may be between about 3 ⁇ m and about 5 ⁇ m in diameter.
  • Each capture-antibody microsphere corresponding to one of the biomarker analytes is loaded with the same indicator. In this manner, each capture-antibody microsphere corresponding to a biomarker analyte is uniquely color-coded.
  • the multiplexed immunoassay device further includes three or more biotinylated detection antibodies in which the antigenic determinant of each biotinylated detection antibody is one of the biomarker analytes.
  • the device further includes a plurality of streptaviden proteins complexed with a reporter compound.
  • a reporter compound as defined herein, is an indicator selected to register a change that is distinguishable from the indicators used to mark the capture-antibody microspheres.
  • the concentrations of the sample analytes may be determined by contacting the test sample with a mixture of capture-antigen microspheres corresponding to each sample analyte to be measured.
  • the sample analytes are allowed to form antigen-antibody complexes in which a sample analyte serves as an antigen and a capture antibody attached to the microsphere serves as an antibody. In this manner, the sample analytes are immobilized onto the capture-antigen microspheres.
  • the biotinylated detection antibodies are then added to the test sample and allowed to form antigen-antibody complexes in which the analyte serves as the antigen and the biotinylated detection antibody serves as the antibody.
  • the streptaviden-reporter complex is then added to the test sample and allowed to bind to the biotin moieties of the biotinylated detection antibodies.
  • the antigen-capture microspheres may then be rinsed and filtered.
  • the concentration of each analyte is determined by first measuring the change registered by the indicator compound embedded in the capture-antigen microsphere in order to identify the particular analyte. For each microsphere corresponding to one of the biomarker analytes, the quantity of analyte immobilized on the microsphere is determined by measuring the change registered by the reporter compound attached to the microsphere.
  • the indicator embedded in the microspheres associated with one sample analyte may register an emission of orange light
  • the reporter may register an emission of green light
  • a detector device may measure the intensity of orange light and green light separately. The measured intensity of the green light would determine the concentration of the analyte captured on the microsphere, and the intensity of the orange light would determine the specific analyte captured on the microsphere.
  • Any sensor device may be used to detect the changes registered by the indicators embedded in the microspheres and the changes registered by the reporter compound, so long as the sensor device is sufficiently sensitive to the changes registered by both indicator and reporter compound.
  • suitable sensor devices include spectrophotometers, photosensors, colorimeters, cyclic coulometry devices, and flow cytometers.
  • the sensor device is a flow cytometer.
  • the multiplexed immunoassay device has a vibrational detection format using a MEMS.
  • the immunoassay device uses capture antibodies as previously described.
  • the capture antibodies are attached to a microscopic silicon microcantilever beam structure.
  • the microcantilevers are micromechanical beams that are anchored at one end, such as diving spring boards that can be readily fabricated on silicon wafers and other materials.
  • the microcantilever sensors are physical sensors that respond to surface stress changes due to chemical or biological processes. When fabricated with very small force constants, they can measure forces and stresses with extremely high sensitivity. The very small force constant of a cantilever allows detection not surface stress variation due to the binding of an analyte to the capture antibody on the microcantilever.
  • Binding of the analyte results in a differential surface stress due to adsorption-induced forces, which manifests as a deflection which can be measured.
  • the vibrational detection may be multiplexed.
  • the kit may include any of the devices described herein in addition to a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
  • the collection apparatus may include, but it not limited to urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, aspiration needles, and combinations thereof.
  • LDD least detectable doses
  • LLOQ lower limits of quantitation
  • the concentrations of the analytes were measured using a capture-sandwich assay using antigen-specific antibodies. For each analyte, a range of standard sample dilutions ranging over about four orders of magnitude of analyte concentration were measured using the assay in order to obtain data used to construct a standard dose response curve.
  • the dynamic range for each of the analytes defined herein as the range of analyte concentrations measured to determine its dose response curve, is presented below.
  • a filter-membrane microtiter plate was pre-wetted by adding 100 ⁇ L wash buffer, and then aspirated using a vacuum manifold device. The contents of the wells of the hard-bottom plate were then transferred to the corresponding wells of the filter-membrane plate. All wells of the hard-bottom plate were vacuum-aspirated and the contents were washed twice with 100 ⁇ L of wash buffer. After the second wash, 100 ⁇ L of wash buffer was added to each well, and then the washed microspheres were resuspended with thorough mixing. The plate was then analyzed using a Luminex 100 Analyzer (Luminex Corporation, Austin, Tex., USA). Dose response curves were constructed for each analyte by curve-fitting the median fluorescence intensity (MFI) measured from the assays of diluted standard samples containing a range of analyte concentrations.
  • MFI median fluorescence intensity
  • the least detectable dose was determined by adding three standard deviations to the average of the MFI signal measured for 20 replicate samples of blank standard solution (i.e. standard solution containing no analyte).
  • the MFI signal was converted to an LDD concentration using the dose response curve and multiplied by a dilution factor of 2.
  • the lower limit of quantification defined herein as the point at which the coefficient of variation (CV) for the analyte measured in the standard samples was 30%, was determined by the analysis of the measurements of increasingly diluted standard samples.
  • the standard solution was diluted by 2 fold for 8 dilutions.
  • samples were assayed in triplicate, and the CV of the analyte concentration at each dilution was calculated and plotted as a function of analyte concentration.
  • the LLOQ was interpolated from this plot and multiplied by a dilution factor of 2.
  • the results of this experiment characterized the least detectible dose and the lower limit of quantification for fourteen analytes associated with various renal disorders using a capture-sandwich assay.
  • the analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • A1M alpha-1 microglobulin
  • B2M beta-2 microglobulin
  • calbindin clusterin
  • CTGF cystatin C
  • GST-alpha cystatin C
  • KIM-1 NGAL
  • osteopontin OPN
  • THP TIMP-1
  • TFF-3 vascular endothelialpha
  • the results of this experiment characterized the precision of a capture-sandwich assay for fourteen analytes associated with various renal disorders over a wide range of analyte concentrations.
  • the precision of the assay varied between about 1% and about 15% error within a given run, and between about 5% and about 15% error between different runs.
  • the percent errors summarized in Table 2 provide information concerning random error to be expected in an assay measurement caused by variations in technicians, measuring instruments, and times of measurement.
  • analytes spiked into urine, serum, and plasma samples were assessed by an assay used to measure the concentration of analytes associated with renal disorders.
  • the analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • the concentrations of the analytes in the samples were measured using the methods described in Example 1.
  • the average % recovery was calculated as the proportion of the measurement of analyte spiked into the urine, serum, or plasma sample (observed) to the measurement of analyte spiked into the standard solution (expected).
  • the results of the spike recovery analysis are summarized in Table 5.
  • the sandwich-type assay is reasonably sensitive to the presence of all analytes measured, whether the analytes were measured in standard samples, urine samples, plasma samples, or serum samples.
  • the analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • A1M alpha-1 microglobulin
  • B2M beta-2 microglobulin
  • calbindin clusterin
  • CTGF cystatin C
  • cystatin C GST-alpha
  • KIM-1 NGAL
  • osteopontin OPN
  • THP TIMP-1
  • TFF-3 VEGF
  • Matrix interference was assessed by spiking hemoglobin, bilirubin, and triglyceride into standard analyte samples and measuring analyte concentrations using the methods described in Example 1. A % recovery was determined by calculating the ratio of the analyte concentration measured from the spiked sample (observed) divided by the analyte concentration measured form the standard sample (expected). The results of the matrix interference analysis are summarized in Table 6.
  • analytes spiked into urine, serum, and plasma samples were assessed to assess the ability of analytes spiked into urine, serum, and plasma samples to tolerate freeze-thaw cycles.
  • the analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • A1M alpha-1 microglobulin
  • B2M beta-2 microglobulin
  • calbindin clusterin
  • CTGF cystatin C
  • GST-alpha cystatin C
  • KIM-1 NGAL
  • osteopontin osteopontin
  • THP TIMP-1
  • TFF-3 VEGF
  • the concentrations of the analytes in the samples were measured using the methods described in Example 1 after the initial addition of the analyte, and after one, two and three cycles of freezing and thawing.
  • analyte concentrations in urine, serum and plasma samples were measured immediately after the addition of the analyte to the samples as well as after storage at room temperature for two hours and four hours, and after storage at 4° C. for 2 hours, four hours, and 24 hours.
  • Clusterin Control 185 100 224 100 171 100 (ng/mL) 2 hr @ 173 94 237 106 180 105 room temp 2 hr. @ 146 79 225 100 171 100 4° C. 4 hr @ 166 89 214 96 160 94 room temp 4 hr. @ 157 85 198 88 143 84 4° C. 24 hr. @ 185 100 207 92 162 94 4° C.
  • CTGF Control 1.9 100 8.8 100 1.2 100 (ng/mL) 2 hr @ 1.9 99 6.7 76 1 83 room temp 2 hr. @ 1.8 96 8.1 92 1.1 89 4° C.
  • KIM-1 Control 1.5 100 0.23 100 0.24 100 (ng/mL) 2 hr @ 1.2 78 0.2 86 0.22 90 room temp 2 hr. @ 1.6 106 0.23 98 0.21 85 4° C. 4 hr @ 1.3 84 0.19 82 0.2 81 room temp 4 hr. @ 1.4 90 0.22 93 0.19 80 4° C. 24 hr. @ 1.1 76 0.18 76 0.23 94 4° C.
  • VEGF Control 851 100 1215 100 670 100 (pg/mL) 2 hr @ 793 93 1055 87 622 93 room temp 2 hr. @ 700 82 1065 88 629 94 4° C.
  • Cystatin C Control 52 100 819 100 476 100 (ng/mL) 2 hr @ 50 96 837 102 466 98 room temp 2 hr. @ 44 84 884 108 547 115 4° C. 4 hr @ 49 93 829 101 498 105 room temp 4 hr. @ 46 88 883 108 513 108 4° C. 24 hr. @ 51 97 767 94 471 99 4° C.
  • NGAL Control 857 100 302 100 93 100 (ng/mL) 2 hr @ 888 104 287 95 96 104 room temp 2 hr. @ 923 108 275 91 92 100 4° C.
  • Urine concentrations of analytes included in a human kidney toxicity panel were measured by the assay, including alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • FIG. 1 summarizes the urine concentrations of those analytes that differed significantly from control urine concentrations.
  • the urine concentrations of A1M, NGAL, and THP were slightly elevated for the renal cancer patient group and more significantly elevated for the “other” cancer patient group.
  • Urine B2M concentrations appeared to be elevated for both the renal cancer and “other” cancer patient groups, although the BRM concentrations exhibited more variability than the other analyte concentrations shown in FIG. 1 .
  • a screen for potential protein biomarkers in relation to kidney toxicity/damage was performed using a panel of biomarkers, in a set of urine and plasma samples from patients with documented renal damage.
  • the investigated patient groups included diabetic nephropathy (DN), obstructive uropathy (OU), analgesic abuse (AA) and glomerulonephritis (GN) along with age, gender and BMI matched control groups.
  • DN diabetic nephropathy
  • OU obstructive uropathy
  • AA analgesic abuse
  • GN glomerulonephritis
  • Multiplexed immunoassays were applied in order to quantify the following protein analytes: Alpha-1 Microglobulin ( ⁇ 1M), KIM-1, Microalbumin, Beta-2-Microglobulin ( ⁇ 32M), Calbindin, Clusterin, CystatinC, TreFoilFactor-3 (TFF-3), CTGF, GST-alpha, VEGF, Calbindin, Osteopontin, Tamm-HorsfallProtein (THP), TIMP-1 and NGAL.
  • Li-Heparin plasma and mid-stream spot urine samples were collected from four different patient groups. Samples were also collected from age, gender and BMI matched control subjects. 20 subjects were included in each group resulting in a total number of 160 urine and plasma samples. All samples were stored at ⁇ 80° C. before use. Glomerular filtration rate for all samples was estimated using two different estimations (Modification of Diet in Renal Disease or MDRD, and the Chronic Kidney Disease Epidemiology Collaboration or CKD-EPI) to outline the eGFR (estimated glomerular filtration rate) distribution within each patient group ( FIG. 2 ). Protein analytes were quantified in human plasma and urine using multiplexed immunoassays in the Luminex xMAPTM platform.
  • microsphere-based multiplex immunoassays consist of antigen-specific antibodies and optimized reagents in a capture-sandwich format. Output data was given as g/ml calculated from internal standard curves. Because urine creatinine (uCr) correlates with renal filtration rate, data analysis was performed without correction for uCr. Univariate and multivariate data analysis was performed comparing all case vs. control samples as well as cases vs. control samples for the various disease groups.
  • Urine and plasma samples were taken from 80 normal control group subjects and 20 subjects from each of four disorders: analgesic abuse, diabetic nephropathy, glomerulonephritis, and obstructive uropathy.
  • the samples were analyzed for the quantity and presence of 16 different proteins (alpha-1 microglobulin ( ⁇ 1M), beta-2 microglobulin ( ⁇ 2M), calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) as described in Example 1 above.
  • the goal was to determine the analytes that distinguish between a normal sample and a diseased sample, a normal sample and an obstructive uropathy (OU) sample, and finally, an glomerulonephritis sample from the other disease samples (diabetic nephropathy (DN), analgesic abuse (AA), and glomerulonephritis (GN)).
  • DN diabetic nephropathy
  • AA analgesic abuse
  • GN glomerulonephritis
  • the mean error rates and AUROC were calculated from urine and AUROC was calculated from plasma for 100 runs of the above method for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal ( FIG. 5 , Table 11), AA vs. normal ( FIG. 7 , Table 13), DN vs. AA ( FIG. 9 , Table 15, AA vs. GN ( FIG. 11 , Table 17), and AA vs. OU ( FIG. 13 , Table 19).

Abstract

Devices for diagnosing, monitoring, or determining a renal disorder in a mammal are described. In particular, devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal are described.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application claims the priority of U.S. provisional application Ser. No. 61/327,389, filed Apr. 23, 2010, and U.S. provisional application Ser. No. 61/232,091, filed Aug. 7, 2009, each of which is hereby incorporated by reference in its entirety, and is related to U.S. patent application Ser. Nos. [Not Yet Assigned], entitled Methods and Devices for Detecting Obstructive Uropathy and Associated Disorders, Computer Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Transplant Rejection, Methods and Devices for Detecting Diabetic Nephropathy and Associated Disorders, and Methods and Devices for Detecting Glomerulonephritis and Associated Disorders, Attorney Docket Nos. 060075-, filed on the same date as this application, the entire contents of which are incorporated herein by reference.
    • FIELD OF THE INVENTION
  • The invention encompasses devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining renal disorders in a mammal using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.
    • BACKGROUND OF THE INVENTION
  • The urinary system, in particular the kidneys, perform several critical functions such as maintaining electrolyte balance and eliminating toxins from the bloodstream. In the human body, the pair of kidneys together process roughly 20% of the total cardiac output, amounting to about 1 L/min in a 70-kg adult male. Because compounds in circulation are concentrated in the kidney up to 1000-fold relative to the plasma concentration, the kidney is especially vulnerable to injury due to exposure to toxic compounds.
  • Renal disorders and disease are serious conditions that generally affect the function of the kidney. The disorders discussed herein may arise from a variety of causes, including intrinsic disease processes, such as inflammation and necrosis of the kidney. In addition, renal disorders may also arise from secondary sources including drugs that are toxic to the kidneys and alternative disease states that cause secondary adverse effects on the kidney, such as diabetes and hypertension. Prevention of renal disorders is largely dependent on early diagnosis of the condition. Existing diagnostic tests such as BUN and serum creatine tests typically detect only advanced stages of kidney damage. Other diagnostic tests such as kidney tissue biopsies or CAT scans have the advantage of enhanced sensitivity to earlier stages of kidney damage, but these tests are also generally costly, slow, and/or invasive.
  • A need exists in the art for a fast, simple, reliable, and sensitive method of detecting obstructive uropathy or an associated disorder. In a clinical setting, the early detection of kidney damage would help medical practitioners to diagnose and treat kidney damage more quickly and effectively.
    • SUMMARY OF THE INVENTION
  • The present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.
  • In one aspect, the present invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
  • In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. It is also recognized that the assay device may include combinations of 6, 10, or 16 antibodies with antigenic determinants corresponding to the analytes disclosed herein.
  • In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising: (a) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol; (b) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (c) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (d) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies.
  • In a further aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; and (b) a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
  • In yet another aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having (i) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; (ii) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (iii) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (iv) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies; and (b) a collection apparatus suitable for collecting a sample bodily fluid from the mammal.
  • In still another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • In a further aspect, the invention encompasses a platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
  • Other aspects and iterations of the invention are described in more detail below.
    • DESCRIPTION OF FIGURES
  • FIG. 1 depicts four graphs comparing (A) the concentrations of alpha-1 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (B) the concentrations of beta-2 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (C) the concentrations of NGAL in the urine of normal controls, kidney cancer patients, and patients with other cancer types; and (D) the concentrations of THP in the urine of normal controls, kidney cancer patients, and patients with other cancer types.
  • FIG. 2 shows the four different disease groups from which samples were analyzed, and a plot of two different estimations on eGFR outlining the distribution within each group.
  • FIG. 3 is a number of scatter plots of results on selected proteins in urine and plasma. The various groups are indicated as follows—control: blue, AA: red, DN: green, GN: yellow, OU: orange. (A) A1M in plasma, (B) cystatin C in plasma, (C) B2M in urine, (D) cystatin C in urine.
  • FIG. 4 depicts the multivariate analysis of the disease groups and their respective matched controls using plasma results. Relative importance shown using the random forest model.
  • FIG. 5 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish disease samples vs. normal samples. Disease encompasses analgesic abuse (AA), glomerulonephritis (GN), obstructive uropathy (OU), and diabetic nephropathy (DN). Normal=NL.
  • FIG. 6 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish disease (AA+GN+ON+DN) samples vs. normal samples from plasma (A) and urine (B and C).
  • FIG. 7 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. normal samples. Abbreviations as in FIG. 4.
  • FIG. 8 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. normal samples from plasma (A) and urine (B and C).
  • FIG. 9 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. diabetic nephropathy samples. Abbreviations as in FIG. 4.
  • FIG. 10 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. diabetic nephropathy samples from plasma (A) and urine (B and C).
  • FIG. 11 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish glomerulonephritis samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.
  • FIG. 12 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish glomerulonephritis samples vs. analgesic abuse samples from plasma (A) and urine (B and C).
  • FIG. 13 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish obstructive uropathy samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.
  • FIG. 14 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish obstructive uropathy samples vs. analgesic abuse samples from plasma (A) and urine (B and C).
    •  DETAILED DESCRIPTION OF THE INVENTION
  • It has been discovered that a multiplexed panel of three or more, six or more, and preferably sixteen, biomarkers may be used to detect renal disorders. As used herein, the term “renal disorder” includes, but is not limited to glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, diabetic nephropathy, analgesic nephropathy, and acute tubular necrosis. As used herein, the term “glomerulonephritis” refers to a disorder characterized by inflammation of the glomeruli. The term may encompass chronic glomerulonephritis, acute glomerulonephritis, primary glomerulonephritis, or secondary glomerulonephritis. As used herein, the term “diabetic nephropathy” refers to a disorder characterized by angiopathy of capillaries in the kidney glomeruli. The term encompasses Kimmelstiel-Wilson syndrome, or nodular diabetic glomerulosclerosis and intercapillary glomerulonephritis. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with diabetic nephropathy. As used herein, the phrase “a disorder associated with diabetic nephropathy” refers to a disorder that stems from angiopathy of capillaries in the kidney glomeruli. For instance, non-limiting examples of associated disorders may include nephritic syndrome, chronic kidney failure, and end-stage kidney disease. The devices of the present invention may also be used to detect secondary kidney damage or toxicity caused by exposure to a toxic compound including but not limited to therapeutic drugs, recreational drugs, medical imaging contrast agents, and toxins. Non-limiting examples of therapeutic drugs may include an analgesic (e.g. aspirin, acetaminophen, ibuprofen, naproxen sodium), an antibiotic (e.g. an aminoglycoside, a beta lactam (cephalosporins, penicillins, penems), rifampin, vancomycin, a sulfonamide, a fluoroquinolone, and a tetracycline), or a chemotherapy agent (e.g. Cisplatin (Platinol®), Carboplatin (Paraplatin®), Cytarabine (Cytosar-U®), Gemtuzumab ozogamicin (Mylotarg®), Gemcitabine (Gemzar®), Melphalan (Alkeran®), Ifosfamide (Ifex®), Methotrexate (Rheumatrex®), Interleukin-2 (Proleukin®), Oxaliplatin (Eloxatin®), Streptozocin (Zanosar®), Pemetrexed (Alimta®), Plicamycin (Mithracin®), and Trimetrexate (Neutrexin®). Further, the term renal disorder may include kidney damage due to kidney stones, ischemia, liver transplantation, heart transplantation, lung transplantation, or hypovolemia. Moreover, the devices of the current invention may be used to detect renal disorders including kidney damage cause by other disease states including but not limited to diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to any infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate disease.
  • In addition, the devices and systems of the current invention may be used to detect renal disorders including acute kidney transplant rejection or chronic allograft nephropathy. Importantly, the devices of the invention may be used to distinguish between an acute rejection reaction and a chronic allograft nephropathy. Alternatively, the devices of the present invention may be used to distinguish between a successful transplant and rejection. As used herein, the term “rejection” refers to a recipient response to a foreign antigen derived from the transplanted kidney. The phrase “acute rejection” refers to an immune related response to the foreign kidney. The response is primarily T-cell driven and originates from an HLC mismatch between the donor and recipient. The phrase “chronic allograft nephropathy” refers to a chronic inflammatory and immune response mediated reaction to a foreign kidney. Chronic allograft nephropathy may result in damage to the kidney manifested by diffuse interstitial fibrosis glomerular changes, typically membranous and sclerotic in nature, as well as intimal fibrosis of the blood vessels with tubular atrophy and loss of tubular structures.
  • Additionally, the present invention encompasses devices comprising biomarkers that may be used to detect a renal disorder associated with kidney transplant rejection. As used herein, the phrase “a disorder associated with kidney transplant rejection” refers to a disorder that stems from a host response to a foreign antigen derived from the transplated kidney. For instance, non-limiting examples of associated disorders may include chronic kidney failure and end-stage kidney disease.
  • The devices of the present invention may also be utilized to detect a renal disorder including obstructive uropathy or an associated disorder in a mammal that includes determining the presence or concentration of a combination of three or more sample analytes in a test sample containing the bodily fluid of the mammal. As used herein, the term “obstructive uropathy” refers to a structural or functional hindrance of normal urine flow. The term may encompass chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, or acute bilateral obstructive uropathy. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with obstructive uropathy. As used herein, the phrase “a disorder associated with obstructive uropathy” refers to a disorder that stems from a structural or functional hindrance of normal urine flow. For instance, non-limiting examples of associated disorders may include hydronephrosis and obstructive nephropathy. The measured concentrations of the combination of sample analytes is compared to the entries of a dataset in which each entry contains the minimum diagnostic concentrations of a combination of three of more analytes reflective of obstructive uropathy or an associated disorder. Other embodiments provide computer-readable media encoded with applications containing executable modules, systems that include databases and processing devices containing executable modules configured to diagnose, monitor, or determine a renal disorder in a mammal. Still other embodiments provide antibody-based devices for diagnosing, monitoring, or determining obstructive uropathy or an associated disorder in a mammal.
  • The biomarkers included in a multiplexed panel of the invention are analytes known in the art that may be detected in the urine, serum, plasma and other bodily fluids of mammals. As such, the analytes of the multiplexed panel may be readily extracted from the mammal in a test sample of bodily fluid. The concentrations of the analytes within the test sample may be measured using known analytical techniques such as a multiplexed antibody-based immunological assay. The combination of concentrations of the analytes in the test sample may be compared to empirically determined combinations of minimum diagnostic concentrations and combinations of diagnostic concentration ranges associated with healthy kidney function to determine whether a renal disorder is indicated in the mammal.
  • The analytes used as biomarkers in the multiplexed assay, methods of diagnosing, monitoring, or determining a renal disorder using measurements of the analytes, systems and applications used to analyze the multiplexed assay measurements, and antibody-based devices used to measure the analytes are described in detail below.
    • I. Analytes in Multiplexed Assay
  • One embodiment of the invention measures the concentrations of three or more, six or more, ten or more, and preferably sixteen, biomarker analytes within a test sample taken from a mammal and compares the measured analyte concentrations to minimum diagnostic concentrations to diagnose, monitor, or determine obstructive uropathy or an associated renal disorder in a mammal. In this aspect, the biomarker analytes are known in the art to occur in the urine, plasma, serum and other bodily fluids of mammals. The biomarker analytes are proteins that have known and documented associations with early renal damage in humans. As defined herein, the biomarker analytes include but are not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. A description of each biomarker analyte is given below. In one embodiment, the biomarker analytes include alpha-1-microglobulin, beta-2-microglobulin, cystatin-C, KIM-1, THP, and TIMP-1.
    • (a) Alpha-1 Microglobulin (A1M)
  • Alpha-1 microglobulin (A1M, Swiss-Prot Accession Number P02760) is a 26 kDa glycoprotein synthesized by the liver and reabsorbed in the proximal tubules. Elevated levels of A1M in human urine are indicative of glomerulotubular dysfunction. A1M is a member of the lipocalin super family and is found in all tissues. Alpha-1-microglobulin exists in blood in both a free form and complexed with immunoglobulin A (IgA) and heme. Half of plasma A1M exists in a free form, and the remainder exists in complexes with other molecules including prothrombin, albumin, immunoglobulin A and heme. Nearly all of the free A1M in human urine is reabsorbed by the megalin receptor in proximal tubular cells, where it is then catabolized. Small amounts of A1M are excreted in the urine of healthy humans. Increased A1M concentrations in human urine may be an early indicator of renal damage, primarily in the proximal tubule.
    • (b) Beta-2 Microglobulin (B2M)
  • Beta-2 microglobulin (B2M, Swiss-Prot Accession Number P61769) is a protein found on the surfaces of all nucleated cells and is shed into the blood, particularly by tumor cells and lymphocytes. Due to its small size, B2M passes through the glomerular membrane, but normally less than 1% is excreted due to reabsorption of B2M in the proximal tubules of the kidney. Therefore, high plasma levels of B2M occur as a result of renal failure, inflammation, and neoplasms, especially those associated with B-lymphocytes.
    • (c) Calbindin
  • Calbindin (Calbindin D-28K, Swiss-Prot Accession Number P05937) is a Ca-binding protein belonging to the troponin C superfamily. It is expressed in the kidney, pancreatic islets, and brain. Calbindin is found predominantly in subpopulations of central and peripheral nervous system neurons, in certain epithelial cells involved in Ca2+ transport such as distal tubular cells and cortical collecting tubules of the kidney, and in enteric neuroendocrine cells.
    • (d) Clusterin
  • Clusterin (Swiss-Prot Accession Number P10909) is a highly conserved protein that has been identified independently by many different laboratories and named SGP2, S35-S45, apolipoprotein J, SP-40, 40, ADHC-9, gp80, GPIII, and testosterone-repressed prostate message (TRPM-2). An increase in clusterin levels has been consistently detected in apoptotic heart, brain, lung, liver, kidney, pancreas, and retinal tissue both in vivo and in vitro, establishing clusterin as a ubiquitous marker of apoptotic cell loss. However, clusterin protein has also been implicated in physiological processes that do not involve apoptosis, including the control of complement-mediated cell lysis, transport of beta-amyloid precursor protein, shuttling of aberrant beta-amyloid across the blood-brain barrier, lipid scavenging, membrane remodeling, cell aggregation, and protection from immune detection and tumor necrosis factor induced cell death.
    • (e) Connective Tissue Growth Factor (CTGF)
  • Connective tissue growth factor (CTGF, Swiss-Prot Accession Number P29279) is a 349-amino acid cysteine-rich polypeptide belonging to the CCN family. In vitro studies have shown that CTGF is mainly involved in extracellular matrix synthesis and fibrosis. Up-regulation of CTGF mRNA and increased CTGF levels have been observed in various diseases, including diabetic nephropathy and cardiomyopathy, fibrotic skin disorders, systemic sclerosis, biliary atresia, liver fibrosis and idiopathic pulmonary fibrosis, and nondiabetic acute and progressive glomerular and tubulointerstitial lesions of the kidney. A recent cross-sectional study found that urinary CTGF may act as a progression promoter in diabetic nephropathy.
    • (f) Creatinine
  • Creatinine is a metabolite of creatine phosphate in muscle tissue, and is typically produced at a relatively constant rate by the body. Creatinine is chiefly filtered out of the blood by the kidneys, though a small amount is actively secreted by the kidneys into the urine. Creatinine levels in blood and urine may be used to estimate the creatinine clearance, which is representative of the overall glomerular filtration rate (GFR), a standard measure of renal function. Variations in creatinine concentrations in the blood and urine, as well as variations in the ratio of urea to creatinine concentration in the blood, are common diagnostic measurements used to assess renal function.
    • (g) Cystatin C (Cyst C)
  • Cystatin C (Cyst C, Swiss-Prot Accession Number P01034) is a 13 kDa protein that is a potent inhibitor of the C1 family of cysteine proteases. It is the most abundant extracellular inhibitor of cysteine proteases in testis, epididymis, prostate, seminal vesicles and many other tissues. Cystatin C, which is normally expressed in vascular wall smooth muscle cells, is severely reduced in both atherosclerotic and aneurismal aortic lesions.
    • (h) Epidermal Growth Factor (EGF)
  • Epidermal growth factor (EGF, Swiss-Prot Accession Number P07522) is a small protein that functions as a potent mitogen. EGF promotes cell growth and differentiation, is essential in embryogenesis, and is important in wound healing. It is produced by many normal cell types and is made in large amounts by certain types of tumors.
  • (i) Glutathione S-Transferase alpha (GST-alpha)
  • Glutathione S-transferase alpha (GST-alpha, Swiss-Prot Accession Number P08263) belongs to a family of enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of compounds, including carcinogens, therapeutic drugs, and products of oxidative stress. These enzymes play a key role in the detoxification of such substances.
    • (j) Glutathione S-Transferase mu (GST-mu)
  • Glutathione S-transferase mu (GST-mu, Swiss-Prot Accession Number PO4905) functions in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione. The genes encoding the mu class of enzymes are organized in a gene cluster on chromosome 1 p13.3 and are known to be highly polymorphic. Genetic variations in GST-mu can change a mammal's susceptibility to carcinogens and toxins as well as affect the toxicity and efficacy of certain drugs. Null mutations of this class mu gene have been linked with an increase in a number of cancers.
    • (k) Kidney Injury Molecule-1 (KIM-1)
  • Kidney injury molecule-1 (KIM-1, Swiss-Prot Accession Number Q96D42) is an immunoglobulin superfamily cell-surface protein highly upregulated on the surface of injured kidney epithelial cells. It is also known as TIM-1 (T-cell immunoglobulin mucin domain-1), as it is expressed at low levels by subpopulations of activated T-cells and hepatitis A virus cellular receptor-1 (HAVCR-1). KIM-1 is increased in expression more than any other protein in the injured kidney and is localized predominantly to the apical membrane of the surviving proximal epithelial cells.
    • (l) Microalbumin
  • Albumin is the most abundant plasma protein in humans and other mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. Healthy, normal kidneys typically filter out albumin from the urine. The presence of albumin in the urine may indicate damage to the kidneys. Albumin in the urine may also occur in patients with long-standing diabetes, especially type 1 diabetes. The amount of albumin eliminated in the urine has been used to differentially diagnose various renal disorders. For example, nephrotic syndrome usually results in the excretion of about 3.0 to 3.5 grams of albumin in human urine every 24 hours. Microalbuminuria, in which less than 300 mg of albumin is eliminated in the urine every 24 hours, may indicate the early stages of diabetic nephropathy.
    • (m) Neutrophil Gelatinase-Associated Lipocalin (NGAL)
  • Neutrophil gelatinase-associated lipocalin (NGAL, Swiss-Prot Accession Number P80188) forms a disulfide bond-linked heterodimer with MMP-9. It mediates an innate immune response to bacterial infection by sequestrating iron. Lipocalins interact with many different molecules such as cell surface receptors and proteases, and play a role in a variety of processes such as the progression of cancer and allergic reactions.
    • (n) Osteopontin (OPN)
  • Osteopontin (OPN, Swiss-Prot Accession Number P10451) is a cytokine involved in enhancing production of interferon-gamma and IL-12, and inhibiting the production of IL-10. OPN is essential in the pathway that leads to type I immunity. OPN appears to form an integral part of the mineralized matrix. OPN is synthesized within the kidney and has been detected in human urine at levels that may effectively inhibit calcium oxalate crystallization. Decreased concentrations of OPN have been documented in urine from patients with renal stone disease compared with normal individuals.
    • (o) Tamm-Horsfall Protein (THP)
  • Tamm-Horsfall protein (THP, Swiss-Prot Accession Number P07911), also known as uromodulin, is the most abundant protein present in the urine of healthy subjects and has been shown to decrease in individuals with kidney stones. THP is secreted by the thick ascending limb of the loop of Henley. THP is a monomeric glycoprotein of ˜85 kDa with ˜30% carbohydrate moiety that is heavily glycosylated. THP may act as a constitutive inhibitor of calcium crystallization in renal fluids.
    • (p) Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)
  • Tissue inhibitor of metalloproteinase-1 (TIMP-1, Swiss-Prot Accession Number P01033) is a major regulator of extracellular matrix synthesis and degradation. A certain balance of MMPs and TIMPs is essential for tumor growth and health. Fibrosis results from an imbalance of fibrogenesis and fibrolysis, highlighting the importance of the role of the inhibition of matrix degradation role in renal disease.
    • (q) Trefoil Factor 3 (TFF3)
  • Trefoil factor 3 (TFF3, Swiss-Prot Accession Number Q07654), also known as intestinal trefoil factor, belongs to a small family of mucin-associated peptides that include TFF1, TFF2, and TFF3. TFF3 exists in a 60-amino acid monomeric form and a 118-amino acid dimeric form. Under normal conditions TFF3 is expressed by goblet cells of the intestine and the colon. TFF3 expression has also been observed in the human respiratory tract, in human goblet cells and in the human salivary gland. In addition, TFF3 has been detected in the human hypothalamus.
    • (r) Vascular Endothelial Growth Factor (VEGF)
  • Vascular endothelial growth factor (VEGF, Swiss-Prot Accession Number P15692) is an important factor in the pathophysiology of neuronal and other tumors, most likely functioning as a potent promoter of angiogenesis. VEGF may also be involved in regulating blood-brain-barrier functions under normal and pathological conditions. VEGF secreted from the stromal cells may be responsible for the endothelial cell proliferation observed in capillary hemangioblastomas, which are typically composed of abundant microvasculature and primitive angiogenic elements represented by stromal cells.
    • (s) Vascular Endothelial Growth Factor A (VEGF A)
  • Vascular endothelial growth factor A (VEGF A, Swiss-Prot Accession Number Q00731) is a growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. It induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces permeabilization of blood vessles. It is important in the pathophysiology of neuronal and other tumors, likely functioning as a potent promoter of angiogenesis. Due to its influences on vascular permeability, VEGF A may be involved in altering blood-brain-barrier functions under normal and pathological conditions. The production and secretion of VEGF by mammalian retinal pigment epithelial cells may be important in the pathogenesis of ocular neovascularization.
    • (t) B-lymphocyte Chemoattractant (BLC)
  • B-lymphocyte chemoattractant (BLC, Swiss-Prot Accession Number 043927) is also referred to as C-X-C motif chemokine 13, Small-inducible cytokine B13, B lymphocyte chemoattractant, CXC chemokine BLC, and B cell-attracting chemokine 1. BLC functions as a potent chemoattractant for B lymphocytes, but not T lymphocytes, monocytes, or neutrophils. Its specific receptor BLR1 is a G protein-coupled receptor originally isolated from Burkitt's lymphoma cells. Among cells of the hematopoietic lineages, the expression of BRL1, now designated CXCR5, is restricted to B lymphocytes and a subpopulation of T helper memory cells.
    • (u) Cluster of Differentiation Surface Receptors 40 (CD40)
  • Cluster of Differentiation Surface Receptors 40 (CD40, Swiss Prot Accession Number P25942) is also referred to TNFRSF5 (Tumor necrosis factor receptor superfamily member 5. CD40 is a member of the tumor necrosis factor-receptor superfamily of proteins. CD40 has been found to be essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.
    • (v) Insulin-Like Growth Factor Binding Protein 2 (IGF BP2)
  • Insulin-like Growth Factor Binding Protein 2 (IGF BP2, Swiss Prot Accession Number P18065) functions to prolong the half-life of the insulin growth factors and have been shown to either inhibit or stimulate the growth promoting effects of the insulin growth factors on cell culture. Specifically, during development, insulin-like growth factor binding protein-2 is expressed in a number of tissues with the highest expression level found in the central nervous system. IGFBP-2 exhibits a 2-10 fold higher affinity for IGF II than for IGF I.
    • (w) Matrix Metalloproteinase-3 (MMP3)
  • Matrix Metalloproteinase-3 (MMP3, Swiss Prot Accession Number P08254) is also known as stromelysin-1 and Transin-1. MMP3 is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. MMP3 encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. The enzyme is thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation. MMP3 is part of a cluster of MMP genes which localize to chromosome 11q22.3.
    • (x) Peptide YY (PYY)
  • Peptide YY (PYY, Swiss-Prot Accession Number P10082) is also known as peptide tyrosine tyrosine and pancreatic peptide YY3-36. Peptide YY exerts its action through neuropeptide Y receptors, inhibits gastric motility and increases water and electrolyte absorption in the colon. PYY may also suppress pancreatic secretion. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite. PYY works by slowing the gastric emptying; hence, it increases efficiency of digestion and nutrient absorption after meal. Research has also indicated that PYY may be useful in removing aluminum accumulated in the brain.
    • (y) Stem Cell Factor (SCF)
  • Stem Cell Factor (SCF, UniProtKB/TrEMBL Q13528) is also known as kit-ligand, KL, and steel factor. SCF functions SCF plays an important role in the hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and decreased pigmentation. During development, the presence of the SCF also plays an important role in the localization of melanocytes, cells that produce melanin and control pigmentation. In melanogenisis, melanoblasts migrate from the neural crest to their appropriate locations in the epidermis. Melanoblasts express the Kit receptor, and it is believed that SCF guides these cells to their terminal locations. SCF also regulates survival and proliferation of fully differentiated melanocytes in adults. In spermatogenesis, c-Kit is expressed in primordial germ cells, spermatogonia, and in primordial oocytes. It is also expressed in the primordial germ cells of females. SCF is expressed along the pathways that the germ cells use to reach their terminal destination in the body. It is also expressed in the final destinations for these cells. Like for melanoblasts, this helps guide the cells to their appropriate locations in the body
    • (z) Tumor Necrosis Factor Receptor Type II (TNF RII)
  • Tumor Necrosis Factor Receptor Type II (TNF RII, Swiss-Prot Accession Number P20333) is also known as p75, p80 TNF alpha receptor, and TNFRSF1B. TNF RII is a protein that in humans is encoded by the TNFRSF1B gene. The protein encoded by this gene is a member of the Tumor necrosis factor receptor superfamily, which also contains TNFRSF1A. The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. The function of IAPs in TNF-receptor signaling is unknown; however, c-IAP1 is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2, which mediates anti-apoptotic signals. Knockout studies in mice also suggest a role of this protein in protecting neurons from apoptosis by stimulating antioxidative pathways.
    • (aa) AXL Oncogene
  • AXL (Swiss-Prot Accession Number P30530) is also known as UFO, ARK, and tyrosine-protein kinase receptor UFO. The protein encoded by AXL is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the AXL protein represents a unique structure of the extracellular region that juxtaposes IgL and FNIII repeats. AXL transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin K-dependent protein growth-arrest-specific gene 6. It is involved in the stimulation of cell proliferation. This receptor can also mediate cell aggregation by homophilic binding. AXL is a chronic myelogenous leukemia-associated oncogene and also associated with colon cancer and melanoma.
    • (bb) Eotaxin 3
  • Eotaxin 3 (Swiss-Prot Accession Number P51671) is also known as C-C motif chemokine 11 (CCL11), small inducible cytokine A11, and eosinophil chemotactic protein. Eotaxin 3 is a small cytokine belonging to the CC chemokine family that is also called Eotaxin-3, Macrophage inflammatory protein 4-alpha (MIP-4-alpha), Thymic stroma chemokine-1 (TSC-1), and IMAC. It is expressed by several tissues including heart, lung and ovary, and in endothelial cells that have been stimulated with the cytokine interleukin 4.[1][2] CCL26 is chemotactic for eosinophils and basophils and elicits its effects by binding to the cell surface chemokine receptor CCR3.
    • (cc) Fatty Acid Binding Protein (FABP)
  • Fatty Acid Binding Protein (FABP, Swiss-Prot Accession Number Q01469) is also known as epidermal-type fatty acid binding protein, and fatty-acid binding protein 5. This gene encodes the fatty acid binding protein found in epidermal cells, and was first identified as being upregulated in psoriasis tissue. Fatty acid binding proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism.
  • (dd) Basic Fibroblast Growth Factor (FGF basic)
  • Basic Fibroblast Growth Factor (FGF basic, Swiss-Prot Accession NumberP09038) is also known as heparin-binding growth factor. In normal tissue, basic fibroblast growth factor is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It stays membrane-bound as long as there is no signal peptide. It has been hypothesized that, during both wound healing of normal tissues and tumor development, the action of heparan sulfate-degrading enzymes activates FGF basic, thus mediating the formation of new blood vessels. Additionally, FGF basic is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by bone morphogenetic proteins. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems.
    • (ee) Myoglobin
  • Myoglobin (Swiss-Prot Accession Number P02144) is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may cause acute renal failure. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest pain.
    • (ff) Resistin (RETN)
  • Resistin (RETN, UniProtKB/TrEMBL Q76B53) is theorized to participate in the inflammatory response. Resistin has also been shown to increase transcriptional events leading to an increased expression of several pro-inflammatory cytokines including (but not limited to) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in an NF-KB-mediated fashion. It has also been demonstrated that resistin upregulates intracellular adhesion molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and CCL2, all of which are occupied in chemotactic pathways involved in leukocyte recruitment to sites of infection. Resistin itself can be upregulated by interleukins and also by microbial antigens such as lipopolysaccharide, which are recognized by leukocytes. Taken together, because resistin is reputed to contribute to insulin resistance, results such as those mentioned suggest that resistin may be a link in the well-known association between inflammation and insulin resistance. In fact, recent data have shown positive correlations between obesity, insulin resistance, and chronic inflammation which is believed to be directed in part by resistin signaling.
    • (gg) Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL R3)
  • TRAIL R3 (Swiss-Prot Accession Number P83626 (mouse)) is also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 3, and tumor necrosis factor receptor mouse homolog. TRAIL R3 is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors.
    • (hh) Endothelin 1 (ET1)
  • Endothelin 1 (ET1, UniProtKB/TrEMBL Q6FH53) is also known as EDN1 and EDN1 protein. Endothelin 1 is a protein that constricts blood vessels and raises blood pressure. It is normally kept in balance by other mechanisms, but when over-expressed, it contributes to high blood pressure (hypertension) and heart disease. Endothelin 1 peptides and receptors are implicated in the pathogenesis of a number of disease states, including cancer and heart disease.
    • (ii) Neuronal Cell Adhesion Molecule (NrCAM)
  • Neuronal Cell Adhesion Molecule (NrCAM, UniProtKB/TrEMBL Q14CA1) encodes a neuronal cell adhesion molecule with multiple immunoglobulin-like C2-type domains and fibronectin type-III domains. This ankyrin-binding protein is involved in neuron-neuron adhesion and promotes directional signaling during axonal cone growth. This gene is also expressed in non-neural tissues and may play a general role in cell-cell communication via signaling from its intracellular domain to the actin cytoskeleton during directional cell migration. Allelic variants of this gene have been associated with autism and addiction vulnerability.
    • (jj) Tenascin C (TN-C)
  • Tenascin C (TN-C, UniProt/TrEMBL Q99857) has anti-adhesive properties, causing cells in tissue culture to become rounded after it is added to the medium. One mechanism to explain this may come from its ability to bind to the extracellular matrix glycoprotein fibronectin and block fibronectin's interactions with specific syndecans. The expression of tenascin-C in the stroma of certain tumors is associated with a poor prognosis.
    • (kk) Vascular Cell Adhesion Molecule 1 (VCAM1)
  • Vascular Cell Adhesion Molecule 1 (VCAM1, Swiss-Prot Accession Number P19320) is also known as vascular cell adhesion protein 1. VCAM1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1)) and through stabilization of Messenger RNA (mRNA) (e.g., Interleukin-4 (IL-4)). The promoter region of the VCAM-1 gene contains functional tandem NF-κB (nuclear factor-kappa B) sites. The sustained expression of VCAM-1 lasts over 24 hours. Primarily, the VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or α4β1) of the β1 subfamily of integrins, and for integrin α4β7. VCAM-1 expression has also been observed in other cell types (e.g., smooth muscle cells). It has also been shown to interact with EZR and Moesin. Certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites.
    • (ll) Cortisol
  • Cortisol (Swiss-Prot Accession Number P08185) is also known as corticosteroid-binding globulin, transcortin, and Serpin A6. Cortisol is a steroid hormone or glucocorticoid produced by the adrenal gland. It is released in response to stress, and to a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis, suppress the immune system, and aid in fat, protein and carbohydrate metabolism. It also decreases bone formation. In addition, cortisol can weaken the activity of the immune system. Cortisol prevents proliferation of T-cells by rendering the interleukin-2 producer T-cells unresponsive to interleukin-1 (IL-1), and unable to produce the T-cell growth factor. Cortisol also has a negative feedback effect on interleukin-1. IL-1 must be especially useful in combating some diseases; however, endotoxin bacteria have gained an advantage by forcing the hypothalamus to increase cortisol levels via forcing secretion of CRH hormone, thus antagonizing IL-1 in this case. The suppressor cells are not affected by GRMF, so that the effective set point for the immune cells may be even higher than the set point for physiological processes. It reflects leukocyte redistribution to lymph nodes, bone marrow, and skin.
    • II. Combinations of Analytes Measured by Multiplexed Assay
  • The device for diagnosing, monitoring, or determining a renal disorder involves determining the presence or concentrations of a combination of sample analytes in a test sample. The combinations of sample analytes, as defined herein, are any group of three or more analytes selected from the biomarker analytes, including but not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. In one embodiment, the combination of analytes may be selected to provide a group of analytes associated with renal disorder in a mammal.
  • In one embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, or all sixteen of the sixteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table A.
  • TABLE A
    alpha-1 microglobulin beta-2 microglobulin calbindin
    alpha-1 microglobulin beta-2 microglobulin clusterin
    alpha-1 microglobulin beta-2 microglobulin CTGF
    alpha-1 microglobulin beta-2 microglobulin creatinine
    alpha-1 microglobulin beta-2 microglobulin cystatin C
    alpha-1 microglobulin beta-2 microglobulin GST-alpha
    alpha-1 microglobulin beta-2 microglobulin KIM-1
    alpha-1 microglobulin beta-2 microglobulin microalbumin
    alpha-1 microglobulin beta-2 microglobulin NGAL
    alpha-1 microglobulin beta-2 microglobulin osteopontin
    alpha-1 microglobulin beta-2 microglobulin THP
    alpha-1 microglobulin beta-2 microglobulin TIMP-1
    alpha-1 microglobulin beta-2 microglobulin TFF-3
    alpha-1 microglobulin beta-2 microglobulin VEGF
    alpha-1 microglobulin calbindin clusterin
    alpha-1 microglobulin calbindin CTGF
    alpha-1 microglobulin calbindin creatinine
    alpha-1 microglobulin calbindin cystatin C
    alpha-1 microglobulin calbindin GST-alpha
    alpha-1 microglobulin calbindin KIM-1
    alpha-1 microglobulin calbindin microalbumin
    alpha-1 microglobulin calbindin NGAL
    alpha-1 microglobulin calbindin osteopontin
    alpha-1 microglobulin calbindin THP
    alpha-1 microglobulin calbindin TIMP-1
    alpha-1 microglobulin calbindin TFF-3
    alpha-1 microglobulin calbindin VEGF
    alpha-1 microglobulin clusterin CTGF
    alpha-1 microglobulin clusterin creatinine
    alpha-1 microglobulin clusterin cystatin C
    alpha-1 microglobulin clusterin GST-alpha
    alpha-1 microglobulin clusterin KIM-1
    alpha-1 microglobulin clusterin microalbumin
    alpha-1 microglobulin clusterin NGAL
    alpha-1 microglobulin clusterin osteopontin
    alpha-1 microglobulin clusterin THP
    alpha-1 microglobulin clusterin TIMP-1
    alpha-1 microglobulin clusterin TFF-3
    alpha-1 microglobulin clusterin VEGF
    alpha-1 microglobulin CTGF creatinine
    alpha-1 microglobulin CTGF cystatin C
    alpha-1 microglobulin CTGF GST-alpha
    alpha-1 microglobulin CTGF KIM-1
    alpha-1 microglobulin CTGF microalbumin
    alpha-1 microglobulin CTGF NGAL
    alpha-1 microglobulin CTGF osteopontin
    alpha-1 microglobulin CTGF THP
    alpha-1 microglobulin CTGF TIMP-1
    alpha-1 microglobulin CTGF TFF-3
    alpha-1 microglobulin CTGF VEGF
    alpha-1 microglobulin creatinine cystatin C
    alpha-1 microglobulin creatinine GST-alpha
    alpha-1 microglobulin creatinine KIM-1
    alpha-1 microglobulin creatinine microalbumin
    alpha-1 microglobulin creatinine NGAL
    alpha-1 microglobulin creatinine osteopontin
    alpha-1 microglobulin creatinine THP
    alpha-1 microglobulin creatinine TIMP-1
    alpha-1 microglobulin creatinine TFF-3
    alpha-1 microglobulin creatinine VEGF
    alpha-1 microglobulin cystatin C GST-alpha
    alpha-1 microglobulin cystatin C KIM-1
    alpha-1 microglobulin cystatin C microalbumin
    alpha-1 microglobulin cystatin C NGAL
    alpha-1 microglobulin cystatin C osteopontin
    alpha-1 microglobulin cystatin C THP
    alpha-1 microglobulin cystatin C TIMP-1
    alpha-1 microglobulin cystatin C TFF-3
    alpha-1 microglobulin cystatin C VEGF
    alpha-1 microglobulin GST-alpha KIM-1
    alpha-1 microglobulin GST-alpha microalbumin
    alpha-1 microglobulin GST-alpha NGAL
    alpha-1 microglobulin GST-alpha osteopontin
    alpha-1 microglobulin GST-alpha THP
    alpha-1 microglobulin GST-alpha TIMP-1
    alpha-1 microglobulin GST-alpha TFF-3
    alpha-1 microglobulin GST-alpha VEGF
    alpha-1 microglobulin KIM-1 microalbumin
    alpha-1 microglobulin KIM-1 NGAL
    alpha-1 microglobulin KIM-1 osteopontin
    alpha-1 microglobulin KIM-1 THP
    alpha-1 microglobulin KIM-1 TIMP-1
    alpha-1 microglobulin KIM-1 TFF-3
    alpha-1 microglobulin KIM-1 VEGF
    alpha-1 microglobulin microalbumin NGAL
    alpha-1 microglobulin microalbumin osteopontin
    alpha-1 microglobulin microalbumin THP
    alpha-1 microglobulin microalbumin TIMP-1
    alpha-1 microglobulin microalbumin TFF-3
    alpha-1 microglobulin microalbumin VEGF
    alpha-1 microglobulin NGAL osteopontin
    alpha-1 microglobulin NGAL THP
    alpha-1 microglobulin NGAL TIMP-1
    alpha-1 microglobulin NGAL TFF-3
    alpha-1 microglobulin NGAL VEGF
    alpha-1 microglobulin osteopontin THP
    alpha-1 microglobulin osteopontin TIMP-1
    alpha-1 microglobulin osteopontin TFF-3
    alpha-1 microglobulin osteopontin VEGF
    alpha-1 microglobulin THP TIMP-1
    alpha-1 microglobulin THP TFF-3
    alpha-1 microglobulin THP VEGF
    alpha-1 microglobulin TIMP-1 TFF-3
    alpha-1 microglobulin TIMP-1 VEGF
    alpha-1 microglobulin TFF-3 VEGF
    beta-2 microglobulin calbindin clusterin
    beta-2 microglobulin calbindin CTGF
    beta-2 microglobulin calbindin creatinine
    beta-2 microglobulin calbindin cystatin C
    beta-2 microglobulin calbindin GST-alpha
    beta-2 microglobulin calbindin KIM-1
    beta-2 microglobulin calbindin microalbumin
    beta-2 microglobulin calbindin NGAL
    beta-2 microglobulin calbindin osteopontin
    beta-2 microglobulin calbindin THP
    beta-2 microglobulin calbindin TIMP-1
    beta-2 microglobulin calbindin TFF-3
    beta-2 microglobulin calbindin VEGF
    beta-2 microglobulin clusterin CTGF
    beta-2 microglobulin clusterin creatinine
    beta-2 microglobulin clusterin cystatin C
    beta-2 microglobulin clusterin GST-alpha
    beta-2 microglobulin clusterin KIM-1
    beta-2 microglobulin clusterin microalbumin
    beta-2 microglobulin clusterin NGAL
    beta-2 microglobulin clusterin osteopontin
    beta-2 microglobulin clusterin THP
    beta-2 microglobulin clusterin TIMP-1
    beta-2 microglobulin clusterin TFF-3
    beta-2 microglobulin clusterin VEGF
    beta-2 microglobulin CTGF creatinine
    beta-2 microglobulin CTGF cystatin C
    beta-2 microglobulin CTGF GST-alpha
    beta-2 microglobulin CTGF KIM-1
    beta-2 microglobulin CTGF microalbumin
    beta-2 microglobulin CTGF NGAL
    beta-2 microglobulin CTGF osteopontin
    beta-2 microglobulin CTGF THP
    beta-2 microglobulin CTGF TIMP-1
    beta-2 microglobulin CTGF TFF-3
    beta-2 microglobulin CTGF VEGF
    beta-2 microglobulin creatinine cystatin C
    beta-2 microglobulin creatinine GST-alpha
    beta-2 microglobulin creatinine KIM-1
    beta-2 microglobulin creatinine microalbumin
    beta-2 microglobulin creatinine NGAL
    beta-2 microglobulin creatinine osteopontin
    beta-2 microglobulin creatinine THP
    beta-2 microglobulin creatinine TIMP-1
    beta-2 microglobulin creatinine TFF-3
    beta-2 microglobulin creatinine VEGF
    beta-2 microglobulin cystatin C GST-alpha
    beta-2 microglobulin cystatin C KIM-1
    beta-2 microglobulin cystatin C microalbumin
    beta-2 microglobulin cystatin C NGAL
    beta-2 microglobulin cystatin C osteopontin
    beta-2 microglobulin cystatin C THP
    beta-2 microglobulin cystatin C TIMP-1
    beta-2 microglobulin cystatin C TFF-3
    beta-2 microglobulin cystatin C VEGF
    beta-2 microglobulin GST-alpha KIM-1
    beta-2 microglobulin GST-alpha microalbumin
    beta-2 microglobulin GST-alpha NGAL
    beta-2 microglobulin GST-alpha osteopontin
    beta-2 microglobulin GST-alpha THP
    beta-2 microglobulin GST-alpha TIMP-1
    beta-2 microglobulin GST-alpha TFF-3
    beta-2 microglobulin GST-alpha VEGF
    beta-2 microglobulin KIM-1 microalbumin
    beta-2 microglobulin KIM-1 NGAL
    beta-2 microglobulin KIM-1 osteopontin
    beta-2 microglobulin KIM-1 THP
    beta-2 microglobulin KIM-1 TIMP-1
    beta-2 microglobulin KIM-1 TFF-3
    beta-2 microglobulin KIM-1 VEGF
    beta-2 microglobulin microalbumin NGAL
    beta-2 microglobulin microalbumin osteopontin
    beta-2 microglobulin microalbumin THP
    beta-2 microglobulin microalbumin TIMP-1
    beta-2 microglobulin microalbumin TFF-3
    beta-2 microglobulin microalbumin VEGF
    beta-2 microglobulin NGAL osteopontin
    beta-2 microglobulin NGAL THP
    beta-2 microglobulin NGAL TIMP-1
    beta-2 microglobulin NGAL TFF-3
    beta-2 microglobulin NGAL VEGF
    beta-2 microglobulin osteopontin THP
    beta-2 microglobulin osteopontin TIMP-1
    beta-2 microglobulin osteopontin TFF-3
    beta-2 microglobulin osteopontin VEGF
    beta-2 microglobulin THP TIMP-1
    beta-2 microglobulin THP TFF-3
    beta-2 microglobulin THP VEGF
    beta-2 microglobulin TIMP-1 TFF-3
    beta-2 microglobulin TIMP-2 VEGF
    beta-2 microglobulin TFF-3 VEGF
    calbindin clusterin CTGF
    calbindin clusterin creatinine
    calbindin clusterin cystatin C
    calbindin clusterin GST-alpha
    calbindin clusterin KIM-1
    calbindin clusterin microalbumin
    calbindin clusterin NGAL
    calbindin clusterin osteopontin
    calbindin clusterin THP
    calbindin clusterin TIMP-1
    calbindin clusterin TFF-3
    calbindin clusterin VEGF
    calbindin CTGF creatinine
    calbindin CTGF cystatin C
    calbindin CTGF GST-alpha
    calbindin CTGF KIM-1
    calbindin CTGF microalbumin
    calbindin CTGF NGAL
    calbindin CTGF osteopontin
    calbindin CTGF THP
    calbindin CTGF TIMP-1
    calbindin CTGF TFF-3
    calbindin CTGF VEGF
    calbindin creatinine cystatin C
    calbindin creatinine GST-alpha
    calbindin creatinine KIM-1
    calbindin creatinine microalbumin
    calbindin creatinine NGAL
    calbindin creatinine osteopontin
    calbindin creatinine THP
    calbindin creatinine TIMP-1
    calbindin creatinine TFF-3
    calbindin creatinine VEGF
    calbindin cystatin C GST-alpha
    calbindin cystatin C KIM-1
    calbindin cystatin C microalbumin
    calbindin cystatin C NGAL
    calbindin cystatin C osteopontin
    calbindin cystatin C THP
    calbindin cystatin C TIMP-1
    calbindin cystatin C TFF-3
    calbindin cystatin C VEGF
    calbindin GST-alpha KIM-1
    calbindin GST-alpha microalbumin
    calbindin GST-alpha NGAL
    calbindin GST-alpha osteopontin
    calbindin GST-alpha THP
    calbindin GST-alpha TIMP-1
    calbindin GST-alpha TFF-3
    calbindin GST-alpha VEGF
    calbindin KIM-1 microalbumin
    calbindin KIM-1 NGAL
    calbindin KIM-1 osteopontin
    calbindin KIM-1 THP
    calbindin KIM-1 TIMP-1
    calbindin KIM-1 TFF-3
    calbindin KIM-1 VEGF
    calbindin microalbumin NGAL
    calbindin microalbumin osteopontin
    calbindin microalbumin THP
    calbindin microalbumin TIMP-1
    calbindin microalbumin TFF-3
    calbindin microalbumin VEGF
    calbindin NGAL osteopontin
    calbindin NGAL THP
    calbindin NGAL TIMP-1
    calbindin NGAL TFF-3
    calbindin NGAL VEGF
    calbindin osteopontin THP
    calbindin osteopontin TIMP-1
    calbindin osteopontin TFF-3
    calbindin osteopontin VEGF
    calbindin THP TIMP-1
    calbindin THP TFF-3
    calbindin THP VEGF
    calbindin TIMP-1 TFF-3
    calbindin TIMP-1 VEGF
    calbindin TFF-3 VEGF
    clusterin CTGF creatinine
    clusterin CTGF cystatin C
    clusterin CTGF GST-alpha
    clusterin CTGF KIM-1
    clusterin CTGF microalbumin
    clusterin CTGF NGAL
    clusterin CTGF osteopontin
    clusterin CTGF THP
    clusterin CTGF TIMP-1
    clusterin CTGF TFF-3
    clusterin CTGF VEGF
    clusterin creatinine cystatin C
    clusterin creatinine GST-alpha
    clusterin creatinine KIM-1
    clusterin creatinine microalbumin
    clusterin creatinine NGAL
    clusterin creatinine osteopontin
    clusterin creatinine THP
    clusterin creatinine TIMP-1
    clusterin creatinine TFF-3
    clusterin creatinine VEGF
    clusterin cystatin C GST-alpha
    clusterin cystatin C KIM-1
    clusterin cystatin C microalbumin
    clusterin cystatin C NGAL
    clusterin cystatin C osteopontin
    clusterin cystatin C THP
    clusterin cystatin C TIMP-1
    clusterin cystatin C TFF-3
    clusterin cystatin C VEGF
    clusterin GST-alpha KIM-1
    clusterin GST-alpha microalbumin
    clusterin GST-alpha NGAL
    clusterin GST-alpha osteopontin
    clusterin GST-alpha THP
    clusterin GST-alpha TIMP-1
    clusterin GST-alpha TFF-3
    clusterin GST-alpha VEGF
    clusterin KIM-1 microalbumin
    clusterin KIM-1 NGAL
    clusterin KIM-1 osteopontin
    clusterin KIM-1 THP
    clusterin KIM-1 TIMP-1
    clusterin KIM-1 TFF-3
    clusterin KIM-1 VEGF
    clusterin microalbumin NGAL
    clusterin microalbumin osteopontin
    clusterin microalbumin THP
    clusterin microalbumin TIMP-1
    clusterin microalbumin TFF-3
    clusterin microalbumin VEGF
    clusterin NGAL osteopontin
    clusterin NGAL THP
    clusterin NGAL TIMP-1
    clusterin NGAL TFF-3
    clusterin NGAL VEGF
    clusterin osteopontin THP
    clusterin osteopontin TIMP-1
    clusterin osteopontin TFF-3
    clusterin osteopontin VEGF
    clusterin THP TIMP-1
    clusterin THP TFF-3
    clusterin THP VEGF
    clusterin TIMP-1 TFF-3
    clusterin TIMP-1 VEGF
    clusterin TFF-3 VEGF
    CTGF creatinine cystatin C
    CTGF creatinine GST-alpha
    CTGF creatinine KIM-1
    CTGF creatinine microalbumin
    CTGF creatinine NGAL
    CTGF creatinine osteopontin
    CTGF creatinine THP
    CTGF creatinine TIMP-1
    CTGF creatinine TFF-3
    CTGF creatinine VEGF
    CTGF cystatin C GST-alpha
    CTGF cystatin C KIM-1
    CTGF cystatin C microalbumin
    CTGF cystatin C NGAL
    CTGF cystatin C osteopontin
    CTGF cystatin C THP
    CTGF cystatin C TIMP-1
    CTGF cystatin C TFF-3
    CTGF cystatin C VEGF
    CTGF GST-alpha KIM-1
    CTGF GST-alpha microalbumin
    CTGF GST-alpha NGAL
    CTGF GST-alpha osteopontin
    CTGF GST-alpha THP
    CTGF GST-alpha TIMP-1
    CTGF GST-alpha TFF-3
    CTGF GST-alpha VEGF
    CTGF KIM-1 microalbumin
    CTGF KIM-1 NGAL
    CTGF KIM-1 osteopontin
    CTGF KIM-1 THP
    CTGF KIM-1 TIMP-1
    CTGF KIM-1 TFF-3
    CTGF KIM-1 VEGF
    CTGF microalbumin NGAL
    CTGF microalbumin osteopontin
    CTGF microalbumin THP
    CTGF microalbumin TIMP-1
    CTGF microalbumin TFF-3
    CTGF microalbumin VEGF
    CTGF NGAL osteopontin
    CTGF NGAL THP
    CTGF NGAL TIMP-1
    CTGF NGAL TFF-3
    CTGF NGAL VEGF
    CTGF osteopontin THP
    CTGF osteopontin TIMP-1
    CTGF osteopontin TFF-3
    CTGF osteopontin VEGF
    CTGF THP TIMP-1
    CTGF THP TFF-3
    CTGF THP VEGF
    CTGF TIMP-1 TFF-3
    CTGF TIMP-1 VEGF
    CTGF TFF-3 VEGF
    creatinine cystatin C GST-alpha
    creatinine cystatin C KIM-1
    creatinine cystatin C microalbumin
    creatinine cystatin C NGAL
    creatinine cystatin C osteopontin
    creatinine cystatin C THP
    creatinine cystatin C TIMP-1
    creatinine cystatin C TFF-3
    creatinine cystatin C VEGF
    creatinine GST-alpha KIM-1
    creatinine GST-alpha microalbumin
    creatinine GST-alpha NGAL
    creatinine GST-alpha osteopontin
    creatinine GST-alpha THP
    creatinine GST-alpha TIMP-1
    creatinine GST-alpha TFF-3
    creatinine GST-alpha VEGF
    creatinine KIM-1 microalbumin
    creatinine KIM-1 NGAL
    creatinine KIM-1 osteopontin
    creatinine KIM-1 THP
    creatinine KIM-1 TIMP-1
    creatinine KIM-1 TFF-3
    creatinine KIM-1 VEGF
    creatinine microalbumin NGAL
    creatinine microalbumin osteopontin
    creatinine microalbumin THP
    creatinine microalbumin TIMP-1
    creatinine microalbumin TFF-3
    creatinine microalbumin VEGF
    creatinine NGAL osteopontin
    creatinine NGAL THP
    creatinine NGAL TIMP-1
    creatinine NGAL TFF-3
    creatinine NGAL VEGF
    creatinine osteopontin THP
    creatinine osteopontin TIMP-1
    creatinine osteopontin TFF-3
    creatinine osteopontin VEGF
    creatinine THP TIMP-1
    creatinine THP TFF-3
    creatinine THP VEGF
    creatinine TIMP-1 TFF-3
    creatinine TIMP-1 VEGF
    creatinine TFF-3 VEGF
    cystatin C GST-alpha KIM-1
    cystatin C GST-alpha microalbumin
    cystatin C GST-alpha NGAL
    cystatin C GST-alpha osteopontin
    cystatin C GST-alpha THP
    cystatin C GST-alpha TIMP-1
    cystatin C GST-alpha TFF-3
    cystatin C GST-alpha VEGF
    cystatin C KIM-1 microalbumin
    cystatin C KIM-1 NGAL
    cystatin C KIM-1 osteopontin
    cystatin C KIM-1 THP
    cystatin C KIM-1 TIMP-1
    cystatin C KIM-1 TFF-3
    cystatin C KIM-1 VEGF
    cystatin C microalbumin NGAL
    cystatin C microalbumin osteopontin
    cystatin C microalbumin THP
    cystatin C microalbumin TIMP-1
    cystatin C microalbumin TFF-3
    cystatin C microalbumin VEGF
    cystatin C NGAL osteopontin
    cystatin C NGAL THP
    cystatin C NGAL TIMP-1
    cystatin C NGAL TFF-3
    cystatin C NGAL VEGF
    cystatin C osteopontin THP
    cystatin C osteopontin TIMP-1
    cystatin C osteopontin TFF-3
    cystatin C osteopontin VEGF
    cystatin C THP TIMP-1
    cystatin C THP TFF-3
    cystatin C THP VEGF
    cystatin C TIMP-1 TFF-3
    cystatin C TIMP-1 VEGF
    cystatin C TFF-3 VEGF
    GST-alpha KIM-1 microalbumin
    GST-alpha KIM-1 NGAL
    GST-alpha KIM-1 osteopontin
    GST-alpha KIM-1 THP
    GST-alpha KIM-1 TIMP-1
    GST-alpha KIM-1 TFF-3
    GST-alpha KIM-1 VEGF
    GST-alpha microalbumin NGAL
    GST-alpha microalbumin osteopontin
    GST-alpha microalbumin THP
    GST-alpha microalbumin TIMP-1
    GST-alpha microalbumin TFF-3
    GST-alpha microalbumin VEGF
    GST-alpha NGAL osteopontin
    GST-alpha NGAL THP
    GST-alpha NGAL TIMP-1
    GST-alpha NGAL TFF-3
    GST-alpha NGAL VEGF
    GST-alpha osteopontin THP
    GST-alpha osteopontin TIMP-1
    GST-alpha osteopontin TFF-3
    GST-alpha osteopontin VEGF
    GST-alpha THP TIMP-1
    GST-alpha THP TFF-3
    GST-alpha THP VEGF
    GST-alpha TIMP-1 TFF-3
    GST-alpha TIMP-1 VEGF
    GST-alpha TFF-3 VEGF
    KIM-1 microalbumin NGAL
    KIM-1 microalbumin osteopontin
    KIM-1 microalbumin THP
    KIM-1 microalbumin TIMP-1
    KIM-1 microalbumin TFF-3
    KIM-1 microalbumin VEGF
    KIM-1 NGAL osteopontin
    KIM-1 NGAL THP
    KIM-1 NGAL TIMP-1
    KIM-1 NGAL TFF-3
    KIM-1 NGAL VEGF
    KIM-1 osteopontin THP
    KIM-1 osteopontin TIMP-1
    KIM-1 osteopontin TFF-3
    KIM-1 osteopontin VEGF
    KIM-1 THP TIMP-1
    KIM-1 THP TFF-3
    KIM-1 THP VEGF
    KIM-1 TIMP-1 TFF-3
    KIM-1 TIMP-1 VEGF
    KIM-1 TFF-3 VEGF
    microalbumin NGAL osteopontin
    microalbumin NGAL THP
    microalbumin NGAL TIMP-1
    microalbumin NGAL TFF-3
    microalbumin NGAL VEGF
    microalbumin osteopontin THP
    microalbumin osteopontin TIMP-1
    microalbumin osteopontin TFF-3
    microalbumin osteopontin VEGF
    microalbumin THP TIMP-1
    microalbumin THP TFF-3
    microalbumin THP VEGF
    microalbumin TIMP-1 TFF-3
    microalbumin TIMP-1 VEGF
    microalbumin TFF-3 VEGF
    NGAL osteopontin THP
    NGAL osteopontin TIMP-1
    NGAL osteopontin TFF-3
    NGAL osteopontin VEGF
    NGAL THP TIMP-1
    NGAL THP TFF-3
    NGAL THP VEGF
    NGAL TIMP-1 TFF-3
    NGAL TIMP-1 VEGF
    NGAL TFF-3 VEGF
    osteopontin THP TIMP-1
    osteopontin THP TFF-3
    osteopontin THP VEGF
    osteopontin TIMP-1 TFF-3
    osteopontin TIMP-1 VEGF
    osteopontin TFF-3 VEGF
    THP TIMP-1 TFF-3
    THP TIMP-1 VEGF
    THP TFF-3 VEGF
    TIMP-1 TFF-3 VEGF
  • In one exemplary embodiment, the combination of sample analytes may include creatinine, KIM-1, and THP. In another exemplary embodiment, the combination of sample analytes may include microalbumin, creatinine, and KIM-1. In yet another exemplary embodiment, the combination of sample analytes may include creatinine, THP, and A1M. In still another exemplary embodiment, the combination of sample analytes may include microalbumin, TIMP-1, and osteopontin.
  • In still another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of obstructive uropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy include three or more biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy includes six biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin.
  • In yet another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of glomerulonephritis. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephritis include three or more biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephropathy includes six biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
  • In an additional embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of kidney damage or toxicity. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In anotherembodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include three or more biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include six biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
  • In a further embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of diabetic nephropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In another embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include three or more biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include six biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
  • In another embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes discussed previously to diagnose kidney transplant rejection or other associated disease as discussed previously. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, any seventeen, any eighteen, or any nineteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table B.
  • TABLE B
    BLC CD40 IGF BP2
    BLC CD40 MMP3
    BLC CD40 peptide YY
    BLC CD40 stem cell factor
    BLC CD40 TNF RII
    BLC CD40 AXL
    BLC CD40 Eotaxin 3
    BLC CD40 FABP
    BLC CD40 FGF basic
    BLC CD40 myoglobin
    BLC CD40 resistin
    BLC CD40 TRAIL R3
    BLC CD40 endothilin 1
    BLC CD40 NrCAM
    BLC CD40 Tenascin C
    BLC CD40 VCAM1
    BLC CD40 cortisol
    BLC IGF BP2 MMP3
    BLC IGF BP2 peptide YY
    BLC IGF BP2 stem cell factor
    BLC IGF BP2 TNF RII
    BLC IGF BP2 AXL
    BLC IGF BP2 Eotaxin 3
    BLC IGF BP2 FABP
    BLC IGF BP2 FGF basic
    BLC IGF BP2 myoglobin
    BLC IGF BP2 resistin
    BLC IGF BP2 TRAIL R3
    BLC IGF BP2 endothilin 1
    BLC IGF BP2 NrCAM
    BLC IGF BP2 Tenascin C
    BLC IGF BP2 VCAM1
    BLC IGF BP2 cortisol
    BLC MMP3 peptide YY
    BLC MMP3 stem cell factor
    BLC MMP3 TNF RII
    BLC MMP3 AXL
    BLC MMP3 Eotaxin 3
    BLC MMP3 FABP
    BLC MMP3 FGF basic
    BLC MMP3 myoglobin
    BLC MMP3 resistin
    BLC MMP3 TRAIL R3
    BLC MMP3 endothilin 1
    BLC MMP3 NrCAM
    BLC MMP3 Tenascin C
    BLC MMP3 VCAM1
    BLC MMP3 cortisol
    BLC peptide YY stem cell factor
    BLC peptide YY TNF RII
    BLC peptide YY AXL
    BLC peptide YY Eotaxin 3
    BLC peptide YY FABP
    BLC peptide YY FGF basic
    BLC peptide YY myoglobin
    BLC peptide YY resistin
    BLC peptide YY TRAIL R3
    BLC peptide YY endothilin 1
    BLC peptide YY NrCAM
    BLC peptide YY Tenascin C
    BLC peptide YY VCAM1
    BLC peptide YY cortisol
    BLC stem cell factor TNF RII
    BLC stem cell factor AXL
    BLC stem cell factor Eotaxin 3
    BLC stem cell factor FABP
    BLC stem cell factor FGF basic
    BLC stem cell factor myoglobin
    BLC stem cell factor resistin
    BLC stem cell factor TRAIL R3
    BLC stem cell factor endothilin 1
    BLC stem cell factor NrCAM
    BLC stem cell factor Tenascin C
    BLC stem cell factor VCAM1
    BLC stem cell factor cortisol
    BLC TNF RII AXL
    BLC TNF RII Eotaxin 3
    BLC TNF RII FABP
    BLC TNF RII FGF basic
    BLC TNF RII myoglobin
    BLC TNF RII resistin
    BLC TNF RII TRAIL R3
    BLC TNF RII endothilin 1
    BLC TNF RII NrCAM
    BLC TNF RII Tenascin C
    BLC TNF RII VCAM1
    BLC TNF RII cortisol
    BLC AXL Eotaxin 3
    BLC AXL FABP
    BLC AXL FGF basic
    BLC AXL myoglobin
    BLC AXL resistin
    BLC AXL TRAIL R3
    BLC AXL endothilin 1
    BLC AXL NrCAM
    BLC AXL Tenascin C
    BLC AXL VCAM1
    BLC AXL cortisol
    BLC Eotaxin 3 FABP
    BLC Eotaxin 3 FGF basic
    BLC Eotaxin 3 myoglobin
    BLC Eotaxin 3 resistin
    BLC Eotaxin 3 TRAIL R3
    BLC Eotaxin 3 endothilin 1
    BLC Eotaxin 3 NrCAM
    BLC Eotaxin 3 Tenascin C
    BLC Eotaxin 3 VCAM1
    BLC Eotaxin 3 cortisol
    BLC FABP FGF basic
    BLC FABP myoglobin
    BLC FABP resistin
    BLC FABP TRAIL R3
    BLC FABP endothilin 1
    BLC FABP NrCAM
    BLC FABP Tenascin C
    BLC FABP VCAM1
    BLC FABP cortisol
    BLC FGF basic myoglobin
    BLC FGF basic resistin
    BLC FGF basic TRAIL R3
    BLC FGF basic endothilin 1
    BLC FGF basic NrCAM
    BLC FGF basic Tenascin C
    BLC FGF basic VCAM1
    BLC FGF basic cortisol
    BLC myoglobin resistin
    BLC myoglobin TRAIL R3
    BLC myoglobin endothilin 1
    BLC myoglobin NrCAM
    BLC myoglobin Tenascin C
    BLC myoglobin VCAM1
    BLC myoglobin cortisol
    BLC resistin TRAIL R3
    BLC resistin endothilin 1
    BLC resistin NrCAM
    BLC resistin Tenascin C
    BLC resistin VCAM1
    BLC resistin cortisol
    BLC TRAIL R3 endothilin 1
    BLC TRAIL R3 NrCAM
    BLC TRAIL R3 Tenascin C
    BLC TRAIL R3 VCAM1
    BLC TRAIL R3 cortisol
    BLC endothilin 1 NrCAM
    BLC endothilin 1 Tenascin C
    BLC endothilin 1 VCAM1
    BLC endothilin 1 cortisol
    BLC NrCAM Tenascin C
    BLC NrCAM VCAM1
    BLC NrCAM cortisol
    BLC Tenascin C VCAM1
    BLC Tenascin C cortisol
    BLC VCAM1 cortisol
    CD40 IGF BP2 MMP3
    CD40 IGF BP2 peptide YY
    CD40 IGF BP2 stem cell factor
    CD40 IGF BP2 TNF RII
    CD40 IGF BP2 AXL
    CD40 IGF BP2 Eotaxin 3
    CD40 IGF BP2 FABP
    CD40 IGF BP2 FGF basic
    CD40 IGF BP2 myoglobin
    CD40 IGF BP2 resistin
    CD40 IGF BP2 TRAIL R3
    CD40 IGF BP2 endothilin 1
    CD40 IGF BP2 NrCAM
    CD40 IGF BP2 Tenascin C
    CD40 IGF BP2 VCAM1
    CD40 IGF BP2 cortisol
    CD40 MMP3 peptide YY
    CD40 MMP3 stem cell factor
    CD40 MMP3 TNF RII
    CD40 MMP3 AXL
    CD40 MMP3 Eotaxin 3
    CD40 MMP3 FABP
    CD40 MMP3 FGF basic
    CD40 MMP3 myoglobin
    CD40 MMP3 resistin
    CD40 MMP3 TRAIL R3
    CD40 MMP3 endothilin 1
    CD40 MMP3 NrCAM
    CD40 MMP3 Tenascin C
    CD40 MMP3 VCAM1
    CD40 MMP3 cortisol
    CD40 peptide YY stem cell factor
    CD40 peptide YY TNF RII
    CD40 peptide YY AXL
    CD40 peptide YY Eotaxin 3
    CD40 peptide YY FABP
    CD40 peptide YY FGF basic
    CD40 peptide YY myoglobin
    CD40 peptide YY resistin
    CD40 peptide YY TRAIL R3
    CD40 peptide YY endothilin 1
    CD40 peptide YY NrCAM
    CD40 peptide YY Tenascin C
    CD40 peptide YY VCAM1
    CD40 peptide YY cortisol
    CD40 stem cell factor TNF RII
    CD40 stem cell factor AXL
    CD40 stem cell factor Eotaxin 3
    CD40 stem cell factor FABP
    CD40 stem cell factor FGF basic
    CD40 stem cell factor myoglobin
    CD40 stem cell factor resistin
    CD40 stem cell factor TRAIL R3
    CD40 stem cell factor endothilin 1
    CD40 stem cell factor NrCAM
    CD40 stem cell factor Tenascin C
    CD40 stem cell factor VCAM1
    CD40 stem cell factor cortisol
    CD40 TNF RII AXL
    CD40 TNF RII Eotaxin 3
    CD40 TNF RII FABP
    CD40 TNF RII FGF basic
    CD40 TNF RII myoglobin
    CD40 TNF RII resistin
    CD40 TNF RII TRAIL R3
    CD40 TNF RII endothilin 1
    CD40 TNF RII NrCAM
    CD40 TNF RII Tenascin C
    CD40 TNF RII VCAM1
    CD40 TNF RII cortisol
    CD40 AXL Eotaxin 3
    CD40 AXL FABP
    CD40 AXL FGF basic
    CD40 AXL myoglobin
    CD40 AXL resistin
    CD40 AXL TRAIL R3
    CD40 AXL endothilin 1
    CD40 AXL NrCAM
    CD40 AXL Tenascin C
    CD40 AXL VCAM1
    CD40 AXL cortisol
    CD40 Eotaxin 3 FABP
    CD40 Eotaxin 3 FGF basic
    CD40 Eotaxin 3 myoglobin
    CD40 Eotaxin 3 resistin
    CD40 Eotaxin 3 TRAIL R3
    CD40 Eotaxin 3 endothilin 1
    CD40 Eotaxin 3 NrCAM
    CD40 Eotaxin 3 Tenascin C
    CD40 Eotaxin 3 VCAM1
    CD40 Eotaxin 3 cortisol
    CD40 FABP FGF basic
    CD40 FABP myoglobin
    CD40 FABP resistin
    CD40 FABP TRAIL R3
    CD40 FABP endothilin 1
    CD40 FABP NrCAM
    CD40 FABP Tenascin C
    CD40 FABP VCAM1
    CD40 FABP cortisol
    CD40 FGF basic myoglobin
    CD40 FGF basic resistin
    CD40 FGF basic TRAIL R3
    CD40 FGF basic endothilin 1
    CD40 FGF basic NrCAM
    CD40 FGF basic Tenascin C
    CD40 FGF basic VCAM1
    CD40 FGF basic cortisol
    CD40 myoglobin resistin
    CD40 myoglobin TRAIL R3
    CD40 myoglobin endothilin 1
    CD40 myoglobin NrCAM
    CD40 myoglobin Tenascin C
    CD40 myoglobin VCAM1
    CD40 myoglobin cortisol
    CD40 resistin TRAIL R3
    CD40 resistin endothilin 1
    CD40 resistin NrCAM
    CD40 resistin Tenascin C
    CD40 resistin VCAM1
    CD40 resistin cortisol
    CD40 TRAIL R3 endothilin 1
    CD40 TRAIL R3 NrCAM
    CD40 TRAIL R3 Tenascin C
    CD40 TRAIL R3 VCAM1
    CD40 TRAIL R3 cortisol
    CD40 endothilin 1 NrCAM
    CD40 endothilin 1 Tenascin C
    CD40 endothilin 1 VCAM1
    CD40 endothilin 1 cortisol
    CD40 NrCAM Tenascin C
    CD40 NrCAM VCAM1
    CD40 NrCAM cortisol
    CD40 Tenascin C VCAM1
    CD40 Tenascin C cortisol
    CD40 VCAM1 cortisol
    IGF BP2 MMP3 peptide YY
    IGF BP2 MMP3 stem cell factor
    IGF BP2 MMP3 TNF RII
    IGF BP2 MMP3 AXL
    IGF BP2 MMP3 Eotaxin 3
    IGF BP2 MMP3 FABP
    IGF BP2 MMP3 FGF basic
    IGF BP2 MMP3 myoglobin
    IGF BP2 MMP3 resistin
    IGF BP2 MMP3 TRAIL R3
    IGF BP2 MMP3 endothilin 1
    IGF BP2 MMP3 NrCAM
    IGF BP2 MMP3 Tenascin C
    IGF BP2 MMP3 VCAM1
    IGF BP2 MMP3 cortisol
    IGF BP2 peptide YY stem cell factor
    IGF BP2 peptide YY TNF RII
    IGF BP2 peptide YY AXL
    IGF BP2 peptide YY Eotaxin 3
    IGF BP2 peptide YY FABP
    IGF BP2 peptide YY FGF basic
    IGF BP2 peptide YY myoglobin
    IGF BP2 peptide YY resistin
    IGF BP2 peptide YY TRAIL R3
    IGF BP2 peptide YY endothilin 1
    IGF BP2 peptide YY NrCAM
    IGF BP2 peptide YY Tenascin C
    IGF BP2 peptide YY VCAM1
    IGF BP2 peptide YY cortisol
    IGF BP2 stem cell factor TNF RII
    IGF BP2 stem cell factor AXL
    IGF BP2 stem cell factor Eotaxin 3
    IGF BP2 stem cell factor FABP
    IGF BP2 stem cell factor FGF basic
    IGF BP2 stem cell factor myoglobin
    IGF BP2 stem cell factor resistin
    IGF BP2 stem cell factor TRAIL R3
    IGF BP2 stem cell factor endothilin 1
    IGF BP2 stem cell factor NrCAM
    IGF BP2 stem cell factor Tenascin C
    IGF BP2 stem cell factor VCAM1
    IGF BP2 stem cell factor cortisol
    IGF BP2 TNF RII AXL
    IGF BP2 TNF RII Eotaxin 3
    IGF BP2 TNF RII FABP
    IGF BP2 TNF RII FGF basic
    IGF BP2 TNF RII myoglobin
    IGF BP2 TNF RII resistin
    IGF BP2 TNF RII TRAIL R3
    IGF BP2 TNF RII endothilin 1
    IGF BP2 TNF RII NrCAM
    IGF BP2 TNF RII Tenascin C
    IGF BP2 TNF RII VCAM1
    IGF BP2 TNF RII cortisol
    IGF BP2 AXL Eotaxin 3
    IGF BP2 AXL FABP
    IGF BP2 AXL FGF basic
    IGF BP2 AXL myoglobin
    IGF BP2 AXL resistin
    IGF BP2 AXL TRAIL R3
    IGF BP2 AXL endothilin 1
    IGF BP2 AXL NrCAM
    IGF BP2 AXL Tenascin C
    IGF BP2 AXL VCAM1
    IGF BP2 AXL cortisol
    IGF BP2 Eotaxin 3 FABP
    IGF BP2 Eotaxin 3 FGF basic
    IGF BP2 Eotaxin 3 myoglobin
    IGF BP2 Eotaxin 3 resistin
    IGF BP2 Eotaxin 3 TRAIL R3
    IGF BP2 Eotaxin 3 endothilin 1
    IGF BP2 Eotaxin 3 NrCAM
    IGF BP2 Eotaxin 3 Tenascin C
    IGF BP2 Eotaxin 3 VCAM1
    IGF BP2 Eotaxin 3 cortisol
    IGF BP2 FABP FGF basic
    IGF BP2 FABP myoglobin
    IGF BP2 FABP resistin
    IGF BP2 FABP TRAIL R3
    IGF BP2 FABP endothilin 1
    IGF BP2 FABP NrCAM
    IGF BP2 FABP Tenascin C
    IGF BP2 FABP VCAM1
    IGF BP2 FABP cortisol
    IGF BP2 FGF basic myoglobin
    IGF BP2 FGF basic resistin
    IGF BP2 FGF basic TRAIL R3
    IGF BP2 FGF basic endothilin 1
    IGF BP2 FGF basic NrCAM
    IGF BP2 FGF basic Tenascin C
    IGF BP2 FGF basic VCAM1
    IGF BP2 FGF basic cortisol
    IGF BP2 myoglobin resistin
    IGF BP2 myoglobin TRAIL R3
    IGF BP2 myoglobin endothilin 1
    IGF BP2 myoglobin NrCAM
    IGF BP2 myoglobin Tenascin C
    IGF BP2 myoglobin VCAM1
    IGF BP2 myoglobin cortisol
    IGF BP2 resistin TRAIL R3
    IGF BP2 resistin endothilin 1
    IGF BP2 resistin NrCAM
    IGF BP2 resistin Tenascin C
    IGF BP2 resistin VCAM1
    IGF BP2 resistin cortisol
    IGF BP2 TRAIL R3 endothilin 1
    IGF BP2 TRAIL R3 NrCAM
    IGF BP2 TRAIL R3 Tenascin C
    IGF BP2 TRAIL R3 VCAM1
    IGF BP2 TRAIL R3 cortisol
    IGF BP2 endothilin 1 NrCAM
    IGF BP2 endothilin 1 Tenascin C
    IGF BP2 endothilin 1 VCAM1
    IGF BP2 endothilin 1 cortisol
    IGF BP2 NrCAM Tenascin C
    IGF BP2 NrCAM VCAM1
    IGF BP2 NrCAM cortisol
    IGF BP2 Tenascin C VCAM1
    IGF BP2 Tenascin C cortisol
    IGF BP2 VCAM1 cortisol
    MMP3 peptide YY stem cell factor
    MMP3 peptide YY TNF RII
    MMP3 peptide YY AXL
    MMP3 peptide YY Eotaxin 3
    MMP3 peptide YY FABP
    MMP3 peptide YY FGF basic
    MMP3 peptide YY myoglobin
    MMP3 peptide YY resistin
    MMP3 peptide YY TRAIL R3
    MMP3 peptide YY endothilin 1
    MMP3 peptide YY NrCAM
    MMP3 peptide YY Tenascin C
    MMP3 peptide YY VCAM1
    MMP3 peptide YY cortisol
    MMP3 stem cell factor TNF RII
    MMP3 stem cell factor AXL
    MMP3 stem cell factor Eotaxin 3
    MMP3 stem cell factor FABP
    MMP3 stem cell factor FGF basic
    MMP3 stem cell factor myoglobin
    MMP3 stem cell factor resistin
    MMP3 stem cell factor TRAIL R3
    MMP3 stem cell factor endothilin 1
    MMP3 stem cell factor NrCAM
    MMP3 stem cell factor Tenascin C
    MMP3 stem cell factor VCAM1
    MMP3 stem cell factor cortisol
    MMP3 TNF RII AXL
    MMP3 TNF RII Eotaxin 3
    MMP3 TNF RII FABP
    MMP3 TNF RII FGF basic
    MMP3 TNF RII myoglobin
    MMP3 TNF RII resistin
    MMP3 TNF RII TRAIL R3
    MMP3 TNF RII endothilin 1
    MMP3 TNF RII NrCAM
    MMP3 TNF RII Tenascin C
    MMP3 TNF RII VCAM1
    MMP3 TNF RII cortisol
    MMP3 AXL Eotaxin 3
    MMP3 AXL FABP
    MMP3 AXL FGF basic
    MMP3 AXL myoglobin
    MMP3 AXL resistin
    MMP3 AXL TRAIL R3
    MMP3 AXL endothilin 1
    MMP3 AXL NrCAM
    MMP3 AXL Tenascin C
    MMP3 AXL VCAM1
    MMP3 AXL cortisol
    MMP3 Eotaxin 3 FABP
    MMP3 Eotaxin 3 FGF basic
    MMP3 Eotaxin 3 myoglobin
    MMP3 Eotaxin 3 resistin
    MMP3 Eotaxin 3 TRAIL R3
    MMP3 Eotaxin 3 endothilin 1
    MMP3 Eotaxin 3 NrCAM
    MMP3 Eotaxin 3 Tenascin C
    MMP3 Eotaxin 3 VCAM1
    MMP3 Eotaxin 3 cortisol
    MMP3 FABP FGF basic
    MMP3 FABP myoglobin
    MMP3 FABP resistin
    MMP3 FABP TRAIL R3
    MMP3 FABP endothilin 1
    MMP3 FABP NrCAM
    MMP3 FABP Tenascin C
    MMP3 FABP VCAM1
    MMP3 FABP cortisol
    MMP3 FGF basic myoglobin
    MMP3 FGF basic resistin
    MMP3 FGF basic TRAIL R3
    MMP3 FGF basic endothilin 1
    MMP3 FGF basic NrCAM
    MMP3 FGF basic Tenascin C
    MMP3 FGF basic VCAM1
    MMP3 FGF basic cortisol
    MMP3 myoglobin resistin
    MMP3 myoglobin TRAIL R3
    MMP3 myoglobin endothilin 1
    MMP3 myoglobin NrCAM
    MMP3 myoglobin Tenascin C
    MMP3 myoglobin VCAM1
    MMP3 myoglobin cortisol
    MMP3 resistin TRAIL R3
    MMP3 resistin endothilin 1
    MMP3 resistin NrCAM
    MMP3 resistin Tenascin C
    MMP3 resistin VCAM1
    MMP3 resistin cortisol
    MMP3 TRAIL R3 endothilin 1
    MMP3 TRAIL R3 NrCAM
    MMP3 TRAIL R3 Tenascin C
    MMP3 TRAIL R3 VCAM1
    MMP3 TRAIL R3 cortisol
    MMP3 endothilin 1 NrCAM
    MMP3 endothilin 1 Tenascin C
    MMP3 endothilin 1 VCAM1
    MMP3 endothilin 1 cortisol
    MMP3 NrCAM Tenascin C
    MMP3 NrCAM VCAM1
    MMP3 NrCAM cortisol
    MMP3 Tenascin C VCAM1
    MMP3 Tenascin C cortisol
    MMP3 VCAM1 cortisol
    peptide YY stem cell factor TNF RII
    peptide YY stem cell factor AXL
    peptide YY stem cell factor Eotaxin 3
    peptide YY stem cell factor FABP
    peptide YY stem cell factor FGF basic
    peptide YY stem cell factor myoglobin
    peptide YY stem cell factor resistin
    peptide YY stem cell factor TRAIL R3
    peptide YY stem cell factor endothilin 1
    peptide YY stem cell factor NrCAM
    peptide YY stem cell factor Tenascin C
    peptide YY stem cell factor VCAM1
    peptide YY stem cell factor cortisol
    peptide YY TNF RII AXL
    peptide YY TNF RII Eotaxin 3
    peptide YY TNF RII FABP
    peptide YY TNF RII FGF basic
    peptide YY TNF RII myoglobin
    peptide YY TNF RII resistin
    peptide YY TNF RII TRAIL R3
    peptide YY TNF RII endothilin 1
    peptide YY TNF RII NrCAM
    peptide YY TNF RII Tenascin C
    peptide YY TNF RII VCAM1
    peptide YY TNF RII cortisol
    peptide YY AXL Eotaxin 3
    peptide YY AXL FABP
    peptide YY AXL FGF basic
    peptide YY AXL myoglobin
    peptide YY AXL resistin
    peptide YY AXL TRAIL R3
    peptide YY AXL endothilin 1
    peptide YY AXL NrCAM
    peptide YY AXL Tenascin C
    peptide YY AXL VCAM1
    peptide YY AXL cortisol
    peptide YY Eotaxin 3 FABP
    peptide YY Eotaxin 3 FGF basic
    peptide YY Eotaxin 3 myoglobin
    peptide YY Eotaxin 3 resistin
    peptide YY Eotaxin 3 TRAIL R3
    peptide YY Eotaxin 3 endothilin 1
    peptide YY Eotaxin 3 NrCAM
    peptide YY Eotaxin 3 Tenascin C
    peptide YY Eotaxin 3 VCAM1
    peptide YY Eotaxin 3 cortisol
    peptide YY FABP FGF basic
    peptide YY FABP myoglobin
    peptide YY FABP resistin
    peptide YY FABP TRAIL R3
    peptide YY FABP endothilin 1
    peptide YY FABP NrCAM
    peptide YY FABP Tenascin C
    peptide YY FABP VCAM1
    peptide YY FABP cortisol
    peptide YY FGF basic myoglobin
    peptide YY FGF basic resistin
    peptide YY FGF basic TRAIL R3
    peptide YY FGF basic endothilin 1
    peptide YY FGF basic NrCAM
    peptide YY FGF basic Tenascin C
    peptide YY FGF basic VCAM1
    peptide YY FGF basic cortisol
    peptide YY myoglobin resistin
    peptide YY myoglobin TRAIL R3
    peptide YY myoglobin endothilin 1
    peptide YY myoglobin NrCAM
    peptide YY myoglobin Tenascin C
    peptide YY myoglobin VCAM1
    peptide YY myoglobin cortisol
    peptide YY resistin TRAIL R3
    peptide YY resistin endothilin 1
    peptide YY resistin NrCAM
    peptide YY resistin Tenascin C
    peptide YY resistin VCAM1
    peptide YY resistin cortisol
    peptide YY TRAIL R3 endothilin 1
    peptide YY TRAIL R3 NrCAM
    peptide YY TRAIL R3 Tenascin C
    peptide YY TRAIL R3 VCAM1
    peptide YY TRAIL R3 cortisol
    peptide YY endothilin 1 NrCAM
    peptide YY endothilin 1 Tenascin C
    peptide YY endothilin 1 VCAM1
    peptide YY endothilin 1 cortisol
    peptide YY NrCAM Tenascin C
    peptide YY NrCAM VCAM1
    peptide YY NrCAM cortisol
    peptide YY Tenascin C VCAM1
    peptide YY Tenascin C cortisol
    peptide YY VCAM1 cortisol
    stem cell factor TNF RII AXL
    stem cell factor TNF RII Eotaxin 3
    stem cell factor TNF RII FABP
    stem cell factor TNF RII FGF basic
    stem cell factor TNF RII myoglobin
    stem cell factor TNF RII resistin
    stem cell factor TNF RII TRAIL R3
    stem cell factor TNF RII endothilin 1
    stem cell factor TNF RII NrCAM
    stem cell factor TNF RII Tenascin C
    stem cell factor TNF RII VCAM1
    stem cell factor TNF RII cortisol
    stem cell factor AXL Eotaxin 3
    stem cell factor AXL FABP
    stem cell factor AXL FGF basic
    stem cell factor AXL myoglobin
    stem cell factor AXL resistin
    stem cell factor AXL TRAIL R3
    stem cell factor AXL endothilin 1
    stem cell factor AXL NrCAM
    stem cell factor AXL Tenascin C
    stem cell factor AXL VCAM1
    stem cell factor AXL cortisol
    stem cell factor Eotaxin 3 FABP
    stem cell factor Eotaxin 3 FGF basic
    stem cell factor Eotaxin 3 myoglobin
    stem cell factor Eotaxin 3 resistin
    stem cell factor Eotaxin 3 TRAIL R3
    stem cell factor Eotaxin 3 endothilin 1
    stem cell factor Eotaxin 3 NrCAM
    stem cell factor Eotaxin 3 Tenascin C
    stem cell factor Eotaxin 3 VCAM1
    stem cell factor Eotaxin 3 cortisol
    stem cell factor FABP FGF basic
    stem cell factor FABP myoglobin
    stem cell factor FABP resistin
    stem cell factor FABP TRAIL R3
    stem cell factor FABP endothilin 1
    stem cell factor FABP NrCAM
    stem cell factor FABP Tenascin C
    stem cell factor FABP VCAM1
    stem cell factor FABP cortisol
    stem cell factor FGF basic myoglobin
    stem cell factor FGF basic resistin
    stem cell factor FGF basic TRAIL R3
    stem cell factor FGF basic endothilin 1
    stem cell factor FGF basic NrCAM
    stem cell factor FGF basic Tenascin C
    stem cell factor FGF basic VCAM1
    stem cell factor FGF basic cortisol
    stem cell factor myoglobin resistin
    stem cell factor myoglobin TRAIL R3
    stem cell factor myoglobin endothilin 1
    stem cell factor myoglobin NrCAM
    stem cell factor myoglobin Tenascin C
    stem cell factor myoglobin VCAM1
    stem cell factor myoglobin cortisol
    stem cell factor resistin TRAIL R3
    stem cell factor resistin endothilin 1
    stem cell factor resistin NrCAM
    stem cell factor resistin Tenascin C
    stem cell factor resistin VCAM1
    stem cell factor resistin cortisol
    stem cell factor TRAIL R3 endothilin 1
    stem cell factor TRAIL R3 NrCAM
    stem cell factor TRAIL R3 Tenascin C
    stem cell factor TRAIL R3 VCAM1
    stem cell factor TRAIL R3 cortisol
    stem cell factor endothilin 1 NrCAM
    stem cell factor endothilin 1 Tenascin C
    stem cell factor endothilin 1 VCAM1
    stem cell factor endothilin 1 cortisol
    stem cell factor NrCAM Tenascin C
    stem cell factor NrCAM VCAM1
    stem cell factor NrCAM cortisol
    stem cell factor Tenascin C VCAM1
    stem cell factor Tenascin C cortisol
    stem cell factor VCAM1 cortisol
    TNF RII AXL Eotaxin 3
    TNF RII AXL FABP
    TNF RII AXL FGF basic
    TNF RII AXL myoglobin
    TNF RII AXL resistin
    TNF RII AXL TRAIL R3
    TNF RII AXL endothilin 1
    TNF RII AXL NrCAM
    TNF RII AXL Tenascin C
    TNF RII AXL VCAM1
    TNF RII AXL cortisol
    TNF RII Eotaxin 3 FABP
    TNF RII Eotaxin 3 FGF basic
    TNF RII Eotaxin 3 myoglobin
    TNF RII Eotaxin 3 resistin
    TNF RII Eotaxin 3 TRAIL R3
    TNF RII Eotaxin 3 endothilin 1
    TNF RII Eotaxin 3 NrCAM
    TNF RII Eotaxin 3 Tenascin C
    TNF RII Eotaxin 3 VCAM1
    TNF RII Eotaxin 3 cortisol
    TNF RII FABP FGF basic
    TNF RII FABP myoglobin
    TNF RII FABP resistin
    TNF RII FABP TRAIL R3
    TNF RII FABP endothilin 1
    TNF RII FABP NrCAM
    TNF RII FABP Tenascin C
    TNF RII FABP VCAM1
    TNF RII FABP cortisol
    TNF RII FGF basic myoglobin
    TNF RII FGF basic resistin
    TNF RII FGF basic TRAIL R3
    TNF RII FGF basic endothilin 1
    TNF RII FGF basic NrCAM
    TNF RII FGF basic Tenascin C
    TNF RII FGF basic VCAM1
    TNF RII FGF basic cortisol
    TNF RII myoglobin resistin
    TNF RII myoglobin TRAIL R3
    TNF RII myoglobin endothilin 1
    TNF RII myoglobin NrCAM
    TNF RII myoglobin Tenascin C
    TNF RII myoglobin VCAM1
    TNF RII myoglobin cortisol
    TNF RII resistin TRAIL R3
    TNF RII resistin endothilin 1
    TNF RII resistin NrCAM
    TNF RII resistin Tenascin C
    TNF RII resistin VCAM1
    TNF RII resistin cortisol
    TNF RII TRAIL R3 endothilin 1
    TNF RII TRAIL R3 NrCAM
    TNF RII TRAIL R3 Tenascin C
    TNF RII TRAIL R3 VCAM1
    TNF RII TRAIL R3 cortisol
    TNF RII endothilin 1 NrCAM
    TNF RII endothilin 1 Tenascin C
    TNF RII endothilin 1 VCAM1
    TNF RII endothilin 1 cortisol
    TNF RII NrCAM Tenascin C
    TNF RII NrCAM VCAM1
    TNF RII NrCAM cortisol
    TNF RII Tenascin C VCAM1
    TNF RII Tenascin C cortisol
    TNF RII VCAM1 cortisol
    AXL Eotaxin 3 FABP
    AXL Eotaxin 3 FGF basic
    AXL Eotaxin 3 myoglobin
    AXL Eotaxin 3 resistin
    AXL Eotaxin 3 TRAIL R3
    AXL Eotaxin 3 endothilin 1
    AXL Eotaxin 3 NrCAM
    AXL Eotaxin 3 Tenascin C
    AXL Eotaxin 3 VCAM1
    AXL Eotaxin 3 cortisol
    AXL FABP FGF basic
    AXL FABP myoglobin
    AXL FABP resistin
    AXL FABP TRAIL R3
    AXL FABP endothilin 1
    AXL FABP NrCAM
    AXL FABP Tenascin C
    AXL FABP VCAM1
    AXL FABP cortisol
    AXL FGF basic myoglobin
    AXL FGF basic resistin
    AXL FGF basic TRAIL R3
    AXL FGF basic endothilin 1
    AXL FGF basic NrCAM
    AXL FGF basic Tenascin C
    AXL FGF basic VCAM1
    AXL FGF basic cortisol
    AXL myoglobin resistin
    AXL myoglobin TRAIL R3
    AXL myoglobin endothilin 1
    AXL myoglobin NrCAM
    AXL myoglobin Tenascin C
    AXL myoglobin VCAM1
    AXL myoglobin cortisol
    AXL resistin TRAIL R3
    AXL resistin endothilin 1
    AXL resistin NrCAM
    AXL resistin Tenascin C
    AXL resistin VCAM1
    AXL resistin cortisol
    AXL TRAIL R3 endothilin 1
    AXL TRAIL R3 NrCAM
    AXL TRAIL R3 Tenascin C
    AXL TRAIL R3 VCAM1
    AXL TRAIL R3 cortisol
    AXL endothilin 1 NrCAM
    AXL endothilin 1 Tenascin C
    AXL endothilin 1 VCAM1
    AXL endothilin 1 cortisol
    AXL NrCAM Tenascin C
    AXL NrCAM VCAM1
    AXL NrCAM cortisol
    AXL Tenascin C VCAM1
    AXL Tenascin C cortisol
    AXL VCAM1 cortisol
    Eotaxin 3 FABP FGF basic
    Eotaxin 3 FABP myoglobin
    Eotaxin 3 FABP resistin
    Eotaxin 3 FABP TRAIL R3
    Eotaxin 3 FABP endothilin 1
    Eotaxin 3 FABP NrCAM
    Eotaxin 3 FABP Tenascin C
    Eotaxin 3 FABP VCAM1
    Eotaxin 3 FABP cortisol
    Eotaxin 3 FGF basic myoglobin
    Eotaxin 3 FGF basic resistin
    Eotaxin 3 FGF basic TRAIL R3
    Eotaxin 3 FGF basic endothilin 1
    Eotaxin 3 FGF basic NrCAM
    Eotaxin 3 FGF basic Tenascin C
    Eotaxin 3 FGF basic VCAM1
    Eotaxin 3 FGF basic cortisol
    Eotaxin 3 myoglobin resistin
    Eotaxin 3 myoglobin TRAIL R3
    Eotaxin 3 myoglobin endothilin 1
    Eotaxin 3 myoglobin NrCAM
    Eotaxin 3 myoglobin Tenascin C
    Eotaxin 3 myoglobin VCAM1
    Eotaxin 3 myoglobin cortisol
    Eotaxin 3 resistin TRAIL R3
    Eotaxin 3 resistin endothilin 1
    Eotaxin 3 resistin NrCAM
    Eotaxin 3 resistin Tenascin C
    Eotaxin 3 resistin VCAM1
    Eotaxin 3 resistin cortisol
    Eotaxin 3 TRAIL R3 endothilin 1
    Eotaxin 3 TRAIL R3 NrCAM
    Eotaxin 3 TRAIL R3 Tenascin C
    Eotaxin 3 TRAIL R3 VCAM1
    Eotaxin 3 TRAIL R3 cortisol
    Eotaxin 3 endothilin 1 NrCAM
    Eotaxin 3 endothilin 1 Tenascin C
    Eotaxin 3 endothilin 1 VCAM1
    Eotaxin 3 endothilin 1 cortisol
    Eotaxin 3 NrCAM Tenascin C
    Eotaxin 3 NrCAM VCAM1
    Eotaxin 3 NrCAM cortisol
    Eotaxin 3 Tenascin C VCAM1
    Eotaxin 3 Tenascin C cortisol
    Eotaxin 3 VCAM1 cortisol
    FABP FGF basic myoglobin
    FABP FGF basic resistin
    FABP FGF basic TRAIL R3
    FABP FGF basic endothilin 1
    FABP FGF basic NrCAM
    FABP FGF basic Tenascin C
    FABP FGF basic VCAM1
    FABP FGF basic cortisol
    FABP myoglobin resistin
    FABP myoglobin TRAIL R3
    FABP myoglobin endothilin 1
    FABP myoglobin NrCAM
    FABP myoglobin Tenascin C
    FABP myoglobin VCAM1
    FABP myoglobin cortisol
    FABP resistin TRAIL R3
    FABP resistin endothilin 1
    FABP resistin NrCAM
    FABP resistin Tenascin C
    FABP resistin VCAM1
    FABP resistin cortisol
    FABP TRAIL R3 endothilin 1
    FABP TRAIL R3 NrCAM
    FABP TRAIL R3 Tenascin C
    FABP TRAIL R3 VCAM1
    FABP TRAIL R3 cortisol
    FABP endothilin 1 NrCAM
    FABP endothilin 1 Tenascin C
    FABP endothilin 1 VCAM1
    FABP endothilin 1 cortisol
    FABP NrCAM Tenascin C
    FABP NrCAM VCAM1
    FABP NrCAM cortisol
    FABP Tenascin C VCAM1
    FABP Tenascin C cortisol
    FABP VCAM1 cortisol
    FGF basic myoglobin resistin
    FGF basic myoglobin TRAIL R3
    FGF basic myoglobin endothilin 1
    FGF basic myoglobin NrCAM
    FGF basic myoglobin Tenascin C
    FGF basic myoglobin VCAM1
    FGF basic myoglobin cortisol
    FGF basic resistin TRAIL R3
    FGF basic resistin endothilin 1
    FGF basic resistin NrCAM
    FGF basic resistin Tenascin C
    FGF basic resistin VCAM1
    FGF basic resistin cortisol
    FGF basic TRAIL R3 endothilin 1
    FGF basic TRAIL R3 NrCAM
    FGF basic TRAIL R3 Tenascin C
    FGF basic TRAIL R3 VCAM1
    FGF basic TRAIL R3 cortisol
    FGF basic endothilin 1 NrCAM
    FGF basic endothilin 1 Tenascin C
    FGF basic endothilin 1 VCAM1
    FGF basic endothilin 1 cortisol
    FGF basic NrCAM Tenascin C
    FGF basic NrCAM VCAM1
    FGF basic NrCAM cortisol
    FGF basic Tenascin C VCAM1
    FGF basic Tenascin C cortisol
    FGF basic VCAM1 cortisol
    myoglobin resistin TRAIL R3
    myoglobin resistin endothilin 1
    myoglobin resistin NrCAM
    myoglobin resistin Tenascin C
    myoglobin resistin VCAM1
    myoglobin resistin cortisol
    myoglobin TRAIL R3 endothilin 1
    myoglobin TRAIL R3 NrCAM
    myoglobin TRAIL R3 Tenascin C
    myoglobin TRAIL R3 VCAM1
    myoglobin TRAIL R3 cortisol
    myoglobin endothilin 1 NrCAM
    myoglobin endothilin 1 Tenascin C
    myoglobin endothilin 1 VCAM1
    myoglobin endothilin 1 cortisol
    myoglobin NrCAM Tenascin C
    myoglobin NrCAM VCAM1
    myoglobin NrCAM cortisol
    myoglobin Tenascin C VCAM1
    myoglobin Tenascin C cortisol
    myoglobin VCAM1 cortisol
    resistin TRAIL R3 endothilin 1
    resistin TRAIL R3 NrCAM
    resistin TRAIL R3 Tenascin C
    resistin TRAIL R3 VCAM1
    resistin TRAIL R3 cortisol
    resistin endothilin 1 NrCAM
    resistin endothilin 1 Tenascin C
    resistin endothilin 1 VCAM1
    resistin endothilin 1 cortisol
    resistin NrCAM Tenascin C
    resistin NrCAM VCAM1
    resistin NrCAM cortisol
    resistin Tenascin C VCAM1
    resistin Tenascin C cortisol
    resistin VCAM1 cortisol
    TRAIL R3 endothilin 1 NrCAM
    TRAIL R3 endothilin 1 Tenascin C
    TRAIL R3 endothilin 1 VCAM1
    TRAIL R3 endothilin 1 cortisol
    TRAIL R3 NrCAM Tenascin C
    TRAIL R3 NrCAM VCAM1
    TRAIL R3 NrCAM cortisol
    TRAIL R3 Tenascin C VCAM1
    TRAIL R3 Tenascin C cortisol
    TRAIL R3 VCAM1 cortisol
    endothilin 1 NrCAM Tenascin C
    endothilin 1 NrCAM VCAM1
    endothilin 1 NrCAM cortisol
    endothilin 1 Tenascin C VCAM1
    endothilin 1 Tenascin C cortisol
    endothilin 1 VCAM1 cortisol
    NrCAM Tenascin C VCAM1
    NrCAM Tenascin C cortisol
    NrCAM VCAM1 cortisol
    Tenascin C VCAM1 cortisol
    • III. Test Sample
  • The method for diagnosing, monitoring, or determining a renal disorder involves determining the presence of sample analytes in a test sample. A test sample, as defined herein, is an amount of bodily fluid taken from a mammal. Non-limiting examples of bodily fluids include urine, blood, plasma, serum, saliva, semen, perspiration, tears, mucus, and tissue lysates. In an exemplary embodiment, the bodily fluid contained in the test sample is urine, plasma, or serum.
    • (a) Mammals
  • A mammal, as defined herein, is any organism that is a member of the class Mammalia. Non-limiting examples of mammals appropriate for the various embodiments may include humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen. In an exemplary embodiment, the mammal is a human.
  • (b) Devices and Methods of Taking Bodily Fluids from Mammals
  • The bodily fluids of the test sample may be taken from the mammal using any known device or method so long as the analytes to be measured by the multiplexed assay are not rendered undetectable by the multiplexed assay. Non-limiting examples of devices or methods suitable for taking bodily fluid from a mammal include urine sample cups, urethral catheters, swabs, hypodermic needles, thin needle biopsies, hollow needle biopsies, punch biopsies, metabolic cages, and aspiration.
  • In order to adjust the expected concentrations of the sample analytes in the test sample to fall within the dynamic range of the multiplexed assay, the test sample may be diluted to reduce the concentration of the sample analytes prior to analysis. The degree of dilution may depend on a variety of factors including but not limited to the type of multiplexed assay used to measure the analytes, the reagents utilized in the multiplexed assay, and the type of bodily fluid contained in the test sample. In one embodiment, the test sample is diluted by adding a volume of diluent ranging from about ½ of the original test sample volume to about 50,000 times the original test sample volume.
  • In one exemplary embodiment, if the test sample is human urine and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 100 times the original test sample volume prior to analysis. In another exemplary embodiment, if the test sample is human serum and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 5 times the original test sample volume prior to analysis. In yet another exemplary embodiment, if the test sample is human plasma and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 2,000 times the original test sample volume prior to analysis.
  • The diluent may be any fluid that does not interfere with the function of the multiplexed assay used to measure the concentration of the analytes in the test sample. Non-limiting examples of suitable diluents include deionized water, distilled water, saline solution, Ringer's solution, phosphate buffered saline solution, TRIS-buffered saline solution, standard saline citrate, and HEPES-buffered saline.
    • IV. Multiplexed Assay Device
  • In one embodiment, the concentration of a combination of sample analytes is measured using a multiplexed assay device capable of measuring up to 189 of the biomarker analytes. A multiplexed assay device, as defined herein, is an assay capable of simultaneously determining the concentration of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more of the biomarker analytes using a single device and/or method. Any known method of measuring the concentration of the biomarker analytes may be used for the multiplexed assay device. Non-limiting examples of measurement methods suitable for the multiplexed assay device include electrophoresis, mass spectrometry, protein microarrays, surface plasmon resonance, and immunoassays including, but not limited to western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA) methods, vibrational detection using MicroElectroMagnetic Devices (MEMS), and particle-based capture-sandwich immunoassays.
    • (a) Multiplexed Immunoassay Device
  • In one embodiment, the concentrations of the analytes in the test sample are measured using a multiplexed immunoassay device that utilizes capture antibodies marked with indicators to determine the concentration of the sample analytes.
    • (i) Capture Antibodies
  • In the same embodiment, the multiplexed immunoassay device includes three or more capture antibodies. Capture antibodies, as defined herein, are antibodies in which the antigenic determinant is one of the Biomarker Analytes known in the art to have a documented association with early renal damage in humans. The biomarker analytes include, but are note limited to alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. Each of the at least three capture antibodies has a unique antigenic determinant that is one of the biomarker analytes. When contacted with the test sample, the capture antibodies form antigen-antibody complexes in which the analytes serve as antigens.
  • The term “antibody,” as used herein, encompasses a monoclonal ab, an antibody fragment, a chimeric antibody, and a single-chain antibody.
  • In some embodiments, the capture antibodies may be attached to a platform or other substrate having a contact surface in order to immobilize any analytes captured by the capture antibodies. The platform generally incorporates a porous material for immobilizing the analytes. Non-limiting examples of suitable substrates include paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, or plastic strips, beads, or surfaces, such as the inner surface of the well of a microtitration tray. Suitable beads may include polystyrene or latex microspheres.
  • (ii) indicators
  • In one embodiment of the multiplexed immunoassay device, an indicator is attached to each of the three or more capture antibodies. The indicator, as defined herein, is any compound that registers a measurable change to indicate the presence of one of the sample analytes when bound to one of the capture antibodies. Non-limiting examples of indicators include visual indicators and electrochemical indicators.
  • Visual indicators, as defined herein, are compounds that register a change by reflecting a limited subset of the wavelengths of light illuminating the indicator, by fluorescing light after being illuminated, or by emitting light via chemiluminescence. The change registered by visual indicators may be in the visible light spectrum, in the infrared spectrum, or in the ultraviolet spectrum. Non-limiting examples of visual indicators suitable for the multiplexed immunoassay device include nanoparticulate gold, organic particles such as polyurethane or latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, quantum dots, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored or chemiluminescent product.
  • Electrochemical indicators, as defined herein, are compounds that register a change by altering an electrical property. The changes registered by electrochemical indicators may be an alteration in conductivity, resistance, capacitance, current conducted in response to an applied voltage, or voltage required to achieve a desired current. Non-limiting examples of electrochemical indicators include redox species such as ascorbate (vitamin C), vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron and mercury.
  • In this same embodiment, the test sample containing a combination of three or more sample analytes is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as the antigens. After removing any uncomplexed capture antibodies, the concentrations of the three or more analytes are determined by measuring the change registered by the indicators attached to the capture antibodies.
  • In one exemplary embodiment, the indicators are polyurethane or latex microspheres loaded with dye compounds and phycoerythrin.
    • (b) Multiplexed Sandwich Immunoassay Device
  • In another embodiment, the multiplexed immunoassay device has a sandwich assay format. In this embodiment, the multiplexed sandwich immunoassay device includes three or more capture antibodies as previously described. However, in this embodiment, each of the capture antibodies is attached to a capture agent that includes an antigenic moiety. The antigenic moiety serves as the antigenic determinant of a detection antibody, also included in the multiplexed immunoassay device of this embodiment. In addition, an indicator is attached to the detection antibody.
  • In this same embodiment, the test sample is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as antigens. The detection antibodies are then contacted with the test sample and allowed to form antigen-antibody complexes in which the capture agent serves as the antigen for the detection antibody. After removing any uncomplexed detection antibodies the concentration of the analytes are determined by measuring the changes registered by the indicators attached to the detection antibodies.
    • (c) Multiplexing Approaches
  • In the various embodiments of the multiplexed immunoassay devices, the concentrations of each of the sample analytes may be determined using any approach known in the art. In one embodiment, a single indicator compound is attached to each of the three or more antibodies. In addition, each of the capture antibodies having one of the sample analytes as an antigenic determinant is physically separated into a distinct region so that the concentration of each of the sample analytes may be determined by measuring the changes registered by the indicators in each physically separate region corresponding to each of the sample analytes.
  • In another embodiment, each antibody having one of the sample analytes as an antigenic determinant is marked with a unique indicator. In this manner, a unique indicator is attached to each antibody having a single sample analyte as its antigenic determinant. In this embodiment, all antibodies may occupy the same physical space. The concentration of each sample analyte is determined by measuring the change registered by the unique indicator attached to the antibody having the sample analyte as an antigenic determinant.
    • (d) Microsphere-Based Capture-Sandwich Immunoassay Device
  • In an exemplary embodiment, the multiplexed immunoassay device is a microsphere-based capture-sandwich immunoassay device. In this embodiment, the device includes a mixture of three or more capture-antibody microspheres, in which each capture-antibody microsphere corresponds to one of the biomarker analytes. Each capture-antibody microsphere includes a plurality of capture antibodies attached to the outer surface of the microsphere. In this same embodiment, the antigenic determinant of all of the capture antibodies attached to one microsphere is the same biomarker analyte.
  • In this embodiment of the device, the microsphere is a small polystyrene or latex sphere that is loaded with an indicator that is a dye compound. The microsphere may be between about 3 μm and about 5 μm in diameter. Each capture-antibody microsphere corresponding to one of the biomarker analytes is loaded with the same indicator. In this manner, each capture-antibody microsphere corresponding to a biomarker analyte is uniquely color-coded.
  • In this same exemplary embodiment, the multiplexed immunoassay device further includes three or more biotinylated detection antibodies in which the antigenic determinant of each biotinylated detection antibody is one of the biomarker analytes. The device further includes a plurality of streptaviden proteins complexed with a reporter compound. A reporter compound, as defined herein, is an indicator selected to register a change that is distinguishable from the indicators used to mark the capture-antibody microspheres.
  • The concentrations of the sample analytes may be determined by contacting the test sample with a mixture of capture-antigen microspheres corresponding to each sample analyte to be measured. The sample analytes are allowed to form antigen-antibody complexes in which a sample analyte serves as an antigen and a capture antibody attached to the microsphere serves as an antibody. In this manner, the sample analytes are immobilized onto the capture-antigen microspheres. The biotinylated detection antibodies are then added to the test sample and allowed to form antigen-antibody complexes in which the analyte serves as the antigen and the biotinylated detection antibody serves as the antibody. The streptaviden-reporter complex is then added to the test sample and allowed to bind to the biotin moieties of the biotinylated detection antibodies. The antigen-capture microspheres may then be rinsed and filtered.
  • In this embodiment, the concentration of each analyte is determined by first measuring the change registered by the indicator compound embedded in the capture-antigen microsphere in order to identify the particular analyte. For each microsphere corresponding to one of the biomarker analytes, the quantity of analyte immobilized on the microsphere is determined by measuring the change registered by the reporter compound attached to the microsphere.
  • For example, the indicator embedded in the microspheres associated with one sample analyte may register an emission of orange light, and the reporter may register an emission of green light. In this example, a detector device may measure the intensity of orange light and green light separately. The measured intensity of the green light would determine the concentration of the analyte captured on the microsphere, and the intensity of the orange light would determine the specific analyte captured on the microsphere.
  • Any sensor device may be used to detect the changes registered by the indicators embedded in the microspheres and the changes registered by the reporter compound, so long as the sensor device is sufficiently sensitive to the changes registered by both indicator and reporter compound. Non-limiting examples of suitable sensor devices include spectrophotometers, photosensors, colorimeters, cyclic coulometry devices, and flow cytometers. In an exemplary embodiment, the sensor device is a flow cytometer.
    • (e) Vibrational Detection Device
  • In another exemplary embodiment, the multiplexed immunoassay device has a vibrational detection format using a MEMS. In this embodiment, the immunoassay device uses capture antibodies as previously described. However, in this embodiment, the capture antibodies are attached to a microscopic silicon microcantilever beam structure. The microcantilevers are micromechanical beams that are anchored at one end, such as diving spring boards that can be readily fabricated on silicon wafers and other materials. The microcantilever sensors are physical sensors that respond to surface stress changes due to chemical or biological processes. When fabricated with very small force constants, they can measure forces and stresses with extremely high sensitivity. The very small force constant of a cantilever allows detection not surface stress variation due to the binding of an analyte to the capture antibody on the microcantilever. Binding of the analyte results in a differential surface stress due to adsorption-induced forces, which manifests as a deflection which can be measured. The vibrational detection may be multiplexed. For more details, see Datar et al., MRS Bulletin (2009) 34:449-459 and Gaster et al., Nature Medicine (2009) 15:1327-1332, both of which are hereby incorporated by reference in their entireties.
  • It will be understood by one skilled in the art that the devices described herein, as well as all those embodiments within the scope of the current invention may be incorporated into a kit. Generally, the kit may include any of the devices described herein in addition to a collection apparatus suitable for collecting a sample of bodily fluid from the mammal. The collection apparatus may include, but it not limited to urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, aspiration needles, and combinations thereof.
    • EXAMPLES
  • The following examples illustrate various iterations of the invention.
    • Example 1 Least Detectable Dose and Lower Limit of Quantitation of Assay for Analytes Associated with Renal Disorders
  • To assess the least detectable doses (LDD) and lower limits of quantitation (LLOQ) of a variety of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • The concentrations of the analytes were measured using a capture-sandwich assay using antigen-specific antibodies. For each analyte, a range of standard sample dilutions ranging over about four orders of magnitude of analyte concentration were measured using the assay in order to obtain data used to construct a standard dose response curve. The dynamic range for each of the analytes, defined herein as the range of analyte concentrations measured to determine its dose response curve, is presented below.
  • To perform the assay, 5 μL of a diluted mixture of capture-antibody microspheres were mixed with 5 μL of blocker and 10 μL of pre-diluted standard sample in each of the wells of a hard-bottom microtiter plate. After incubating the hard-bottom plate for 1 hour, 10 μL of biotinylated detection antibody was added to each well, and then the hard-bottom plate was incubated for an additional hour. 10 μL of diluted streptavidin-phycoerythrin was added to each well and then the hard-bottom plate was incubated for another 60 minutes.
  • A filter-membrane microtiter plate was pre-wetted by adding 100 μL wash buffer, and then aspirated using a vacuum manifold device. The contents of the wells of the hard-bottom plate were then transferred to the corresponding wells of the filter-membrane plate. All wells of the hard-bottom plate were vacuum-aspirated and the contents were washed twice with 100 μL of wash buffer. After the second wash, 100 μL of wash buffer was added to each well, and then the washed microspheres were resuspended with thorough mixing. The plate was then analyzed using a Luminex 100 Analyzer (Luminex Corporation, Austin, Tex., USA). Dose response curves were constructed for each analyte by curve-fitting the median fluorescence intensity (MFI) measured from the assays of diluted standard samples containing a range of analyte concentrations.
  • The least detectable dose (LDD) was determined by adding three standard deviations to the average of the MFI signal measured for 20 replicate samples of blank standard solution (i.e. standard solution containing no analyte). The MFI signal was converted to an LDD concentration using the dose response curve and multiplied by a dilution factor of 2.
  • The lower limit of quantification (LLOQ), defined herein as the point at which the coefficient of variation (CV) for the analyte measured in the standard samples was 30%, was determined by the analysis of the measurements of increasingly diluted standard samples. For each analyte, the standard solution was diluted by 2 fold for 8 dilutions. At each stage of dilution, samples were assayed in triplicate, and the CV of the analyte concentration at each dilution was calculated and plotted as a function of analyte concentration. The LLOQ was interpolated from this plot and multiplied by a dilution factor of 2.
  • The LDD and LLOQ results for each analyte are summarized in Table 2:
  • TABLE 2
    LDD, LLOQ, and Dynamic Range of Analyte Assay
    Dynamic Range
    Analyte Units LDD LLOQ minimum maximum
    Calbindin ng/mL 1.1 3.1 0.516 2580
    Clusterin ng/mL 2.4 2.3 0.676 3378
    CTGF ng/mL 1.3 3.8 0.0794 400
    GST-alpha ng/mL 1.4 3.6 0.24 1,200
    KIM-1 ng/mL 0.016 0.028 0.00478 24
    VEGF pg/mL 4.4 20 8.76 44,000
    β-2M μg/mL 0.012 0.018 0.0030 15
    Cystatin C ng/mL 2.8 3.7 0.60 3,000
    NGAL ng/mL 4.1 7.8 1.2 6,000
    Osteopontin ng/mL 29 52 3.9 19,500
    TIMP-1 ng/mL 0.71 1.1 0.073 365
    A-1M μg/mL 0.059 0.29 0.042 210
    THP μg/mL 0.46 0.30 0.16 800
    TFF-3 μg/mL 0.06 0.097 0.060 300
  • The results of this experiment characterized the least detectible dose and the lower limit of quantification for fourteen analytes associated with various renal disorders using a capture-sandwich assay.
    • Example 2 Precision of Assay for Analytes Associated with Renal Disorders
  • To assess the precision of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. The percent errors for each run at each concentration are presented in Table 3 for all of the analytes tested:
  • TABLE 3
    Precision of Analyte Assay
    Average Run 2 Interrun
    concentration Run 1 Error Run 2 Error
    Analyte (ng/mL) Error (%) (%) Error (%) (%)
    Calbindin 4.0 6 2 6 13
    36 5 3 2 7
    281 1 6 0 3
    Clusterin 4.4 4 9 2 6
    39 5 1 6 8
    229 1 3 0 2
    CTGF 1.2 10 17 4 14
    2.5 19 19 14 14
    18 7 5 13 9
    GST-alpha 3.9 14 7 5 10
    16 13 7 10 11
    42 1 16 6 8
    KIM-1 0.035 2 0 5 13
    0.32 4 5 2 8
    2.9 0 5 7 4
    VEGF 65 10 1 6 14
    534 9 2 12 7
    5,397 1 13 14 9
    β-2M 0.040 6 1 8 5
    0.43 2 2 0 10
    6.7 6 5 11 6
    Cystatin C 10.5 4 1 7 13
    49 0 0 3 9
    424 2 6 2 5
    NGAL 18.1 11 3 6 13
    147 0 0 6 5
    1,070 5 1 2 5
    Osteopontin 44 1 10 2 11
    523 9 9 9 7
    8,930 4 10 1 10
    TIMP-1 2.2 13 6 3 13
    26 1 1 4 14
    130 1 3 1 4
    A-1M 1.7 11 7 7 14
    19 4 1 8 9
    45 3 5 2 4
    THP 9.4 3 10 11 11
    15 3 7 8 6
    37 4 5 0 5
    TFF-3 0.3 13 3 11 12
    4.2 5 8 5 7
    1.2 3 7 0 13
  • The results of this experiment characterized the precision of a capture-sandwich assay for fourteen analytes associated with various renal disorders over a wide range of analyte concentrations. The precision of the assay varied between about 1% and about 15% error within a given run, and between about 5% and about 15% error between different runs. The percent errors summarized in Table 2 provide information concerning random error to be expected in an assay measurement caused by variations in technicians, measuring instruments, and times of measurement.
    • Example 3 Linearity of Assay for Analytes Associated with Renal Disorders
  • To assess the linearity of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. Linearity of the assay used to measure each analyte was determined by measuring the concentrations of standard samples that were serially-diluted throughout the assay range. The % recovery was calculated as observed vs. expected concentration based on the dose-response curve. The results of the linearity analysis are summarized in Table 4.
  • TABLE 4
    Linearity of Analyte Assay
    Expected Observed Recovery
    Analyte Dilution concentration concentration (%)
    Calbindin 1:2 61 61 100
    (ng/mL) 1:4 30 32 106
    1:8 15 17 110
    Clusterin 1:2 41 41 100
    (ng/mL) 1:4 21 24 116
    1:8 10 11 111
    CTGF 1:2 1.7 1.7 100
    (ng/mL) 1:4 0.84 1.0 124
    1:8 0.42 0.51 122
    GST-alpha 1:2 25 25 100
    (ng/mL) 1:4 12 14 115
    1:8 6.2 8.0 129
    KIM-1 1:2 0.87 0.87 100
    (ng/mL) 1:4 0.41 0.41 101
    1:8 0.21 0.19 93
    VEGF 1:2 2,525 2,525 100
    (pg/mL) 1:4 1,263 1,340 106
    1:8 631 686 109
    β-2M 1:100 0.63 0.63 100
    (μg/mL) 1:200 0.31 0.34 106
    1:400 0.16 0.17 107
    Cystatin C 1:100 249 249 100
    (ng/mL) 1:200 125 122 102
    1:400 62 56 110
    NGAL 1:100 1,435 1,435 100
    (ng/mL) 1:200 718 775 108
    1:400 359 369 103
    Osteopontin 1:100 6,415 6,415 100
    (ng/mL) 1:200 3,208 3,275 102
    1:400 1,604 1,525 95
    TIMP-1 1:100 35 35 100
    (ng/mL) 1:200 18 18 100
    1:400 8.8 8.8 100
    A-1M 1:2000 37 37 100
    (μg/mL) 1:4000 18 18 99
    1:8000 9.1 9.2 99
    THP 1:2000 28 28 100
    (μg/mL) 1:4000 14 14 96
    1:8000 6.7 7.1 94
    TFF-3 1:2000 8.8 8.8 100
    (μg/mL) 1:4000 3.8 4.4 86
    1:8000 1.9 2.2 86
  • The results of this experiment demonstrated reasonably linear responses of the sandwich-capture assay to variations in the concentrations of the analytes in the tested samples.
    • Example 4 Spike Recovery of Analytes Associated with Renal Disorders
  • To assess the recovery of analytes spiked into urine, serum, and plasma samples by an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Prior to analysis, all urine samples were diluted 1:2000 (sample: diluent), all plasma samples were diluted 1:5 (sample: diluent), and all serum samples were diluted 1:2000 (sample: diluent).
  • The concentrations of the analytes in the samples were measured using the methods described in Example 1. The average % recovery was calculated as the proportion of the measurement of analyte spiked into the urine, serum, or plasma sample (observed) to the measurement of analyte spiked into the standard solution (expected). The results of the spike recovery analysis are summarized in Table 5.
  • TABLE 5
    Spike Recovery of Analyte Assay in Urine, Serum, and Plasma Samples
    Recovery in Recovery in Recovery in
    Spike Urine Serum Plasma
    Analyte Concentration Sample (%) Sample (%) Sample (%)
    Calbindin 66 76 82 83
    (ng/mL) 35 91 77 71
    18 80 82 73
    average 82 80 76
    Clusterin 80 72 73 75
    (ng/mL) 37 70 66 72
    20 90 73 70
    average 77 70 72
    CTGF 8.4 91 80 79
    (ng/mL) 4.6 114 69 78
    2.4 76 80 69
    average 94 77 75
    GST-alpha 27 75 84 80
    (ng/mL) 15 90 75 81
    7.1 82 84 72
    average 83 81 78
    KIM-1 0.63 87 80 83
    (ng/mL) .029 119 74 80
    0.14 117 80 78
    average 107 78 80
    VEGF 584 88 84 82
    (pg/mL) 287 101 77 86
    123 107 84 77
    average 99 82 82
    β-2M 0.97 117 98 98
    (μg/mL) 0.50 124 119 119
    0.24 104 107 107
    average 115 108 105
    Cystatin C 183 138 80 103
    (ng/mL) 90 136 97 103
    40 120 97 118
    average 131 91 108
    NGAL 426 120 105 111
    (ng/mL) 213 124 114 112
    103 90 99 113
    average 111 106 112
    Osteopontin 1,245 204 124 68
    (ng/mL) 636 153 112 69
    302 66 103 67
    average 108 113 68
    TIMP-1 25 98 97 113
    (ng/mL) 12 114 89 103
    5.7 94 99 113
    average 102 95 110
    A-1M 0.0028 100 101 79
    (μg/mL) 0.0012 125 80 81
    0.00060 118 101 82
    average 114 94 81
    THP 0.0096 126 108 90
    (μg/mL) 0.0047 131 93 91
    0.0026 112 114 83
    average 123 105 88
    TFF-3 0.0038 105 114 97
    (μg/mL) 0.0019 109 104 95
    0.0010 102 118 93
    average 105 112 95
  • The results of this experiment demonstrated that the sandwich-type assay is reasonably sensitive to the presence of all analytes measured, whether the analytes were measured in standard samples, urine samples, plasma samples, or serum samples.
    • Example 5 Matrix Interferences of Analytes Associated with Renal Disorders
  • To assess the matrix interference of hemoglobin, bilirubin, and triglycerides spiked into standard samples, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Matrix interference was assessed by spiking hemoglobin, bilirubin, and triglyceride into standard analyte samples and measuring analyte concentrations using the methods described in Example 1. A % recovery was determined by calculating the ratio of the analyte concentration measured from the spiked sample (observed) divided by the analyte concentration measured form the standard sample (expected). The results of the matrix interference analysis are summarized in Table 6.
  • TABLE 6
    Matrix Interference of Hemoglobin, Bilirubin, and Triglyceride on
    the Measurement of Analytes
    Matrix
    Compound Maximum Overall
    Spiked into Spike Recovery
    Analyte Sample Concentration (%)
    Calbindin Hemoglobin 500 110
    (mg/mL) Bilirubin 20 98
    Triglyceride 500 117
    Clusterin Hemoglobin 500 125
    (mg/mL) Bilirubin 20 110
    Triglyceride 500 85
    CTGF Hemoglobin 500 91
    (mg/mL) Bilirubin 20 88
    Triglyceride 500 84
    GST-alpha Hemoglobin 500 100
    (mg/mL) Bilirubin 20 96
    Triglyceride 500 96
    KIM-1 Hemoglobin 500 108
    (mg/mL) Bilirubin 20 117
    Triglyceride 500 84
    VEGF Hemoglobin 500 112
    (mg/mL) Bilirubin 20 85
    Triglyceride 500 114
    β-2M Hemoglobin 500 84
    (μg/mL) Bilirubin 20 75
    Triglyceride 500 104
    Cystatin C Hemoglobin 500 91
    (ng/mL) Bilirubin 20 102
    Triglyceride 500 124
    NGAL Hemoglobin 500 99
    (ng/mL) Bilirubin 20 92
    Triglyceride 500 106
    Osteopontin Hemoglobin 500 83
    (ng/mL) Bilirubin 20 86
    Triglyceride 500 106
    TIMP-1 Hemoglobin 500 87
    (ng/mL) Bilirubin 20 86
    Triglyceride 500 93
    A-1M Hemoglobin 500 103
    (μg/mL) Bilirubin 20 110
    Triglyceride 500 112
    THP Hemoglobin 500 108
    (μg/mL) Bilirubin 20 101
    Triglyceride 500 121
    TFF-3 Hemoglobin 500 101
    (μg/mL) Bilirubin 20 101
    Triglyceride 500 110
  • The results of this experiment demonstrated that hemoglobin, bilirubin, and triglycerides, three common compounds found in urine, plasma, and serum samples, did not significantly degrade the ability of the sandwich-capture assay to detect any of the analytes tested.
    • Example 6 Sample Stability of Analytes Associated with Renal Disorders
  • To assess the ability of analytes spiked into urine, serum, and plasma samples to tolerate freeze-thaw cycles, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. Each analyte was spiked into known urine, serum, and plasma samples at a known analyte concentration. The concentrations of the analytes in the samples were measured using the methods described in Example 1 after the initial addition of the analyte, and after one, two and three cycles of freezing and thawing. In addition, analyte concentrations in urine, serum and plasma samples were measured immediately after the addition of the analyte to the samples as well as after storage at room temperature for two hours and four hours, and after storage at 4° C. for 2 hours, four hours, and 24 hours.
  • The results of the freeze-thaw stability analysis are summarized in Table 7. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any freeze-thaw cycles.
  • TABLE 7
    Freeze-Thaw Stability of the Analytes in Urine, Serum, and Plasma
    Period Urine Sample Serum Sample Plasma Sample
    and Recovery Recovery Recovery
    Analyte Temp Concentration (%) Concentration (%) Concentration (%)
    Calbindin Control 212 100 31 100 43 100
    (ng/mL) 1X 221 104 30 96 41 94
    2X 203 96 30 99 39 92
    3X 234 110 30 97 40 93
    Clusterin 0 315 100 232 100 187 100
    (ng/mL) 1X 329 104 227 98 177 95
    2X 341 108 240 103 175 94
    3X 379 120 248 107 183 98
    CTGF 0 6.7 100 1.5 100 1.2 100
    (ng/mL) 1X 7.5 112 1.3 82 1.2 94
    2X 6.8 101 1.4 90 1.2 100
    3X 7.7 115 1.2 73 1.3 107
    GST- 0 12 100 23 100 11 100
    alpha 1X 13 104 24 105 11 101
    (ng/mL) 2X 14 116 21 92 11 97
    3X 14 111 23 100 12 108
    KIM-1 0 1.7 100 0.24 100 0.24 100
    (ng/mL) 1X 1.7 99 0.24 102 0.22 91
    2X 1.7 99 0.22 94 0.19 78
    3X 1.8 107 0.23 97 0.22 93
    VEGF 0 1,530 100 1,245 100 674 100
    (pg/mL) 1X 1,575 103 1,205 97 652 97
    2X 1,570 103 1,140 92 612 91
    3X 1,700 111 1,185 95 670 99
    β-2M 0 0.0070 100 1.2 100 15 100
    (μg/mL) 1X 0.0073 104 1.1 93 14 109
    2X 0.0076 108 1.2 103 15 104
    3X 0.0076 108 1.1 97 13 116
    Cystatin C 0 1,240 100 1,330 100 519 100
    (ng/mL) 1X 1,280 103 1,470 111 584 113
    2X 1,410 114 1,370 103 730 141
    3X 1,420 115 1,380 104 589 113
    NGAL 0 45 100 245 100 84 100
    (ng/mL) 1X 46 102 179 114 94 112
    2X 47 104 276 113 91 108
    3X 47 104 278 113 91 109
    Osteopontin 0 38 100 1.7 100 5.0 100
    (ng/mL) 1X 42 110 1.8 102 5.5 110
    2X 42 108 1.5 87 5.5 109
    3X 42 110 1.3 77 5.4 107
    TIMP-1 0 266 100 220 100 70 100
    (ng/mL) 1X 265 100 220 10 75 108
    2X 255 96 215 98 77 110
    3X 295 111 228 104 76 109
    A-1M 0 14 100 26 100 4.5 100
    (μg/mL) 1X 13 92 25 96 4.2 94
    2X 15 107 25 96 4.3 97
    3X 16 116 23 88 4.0 90
    THP 0 4.6 100 31 100 9.2 100
    (μg/mL) 1X 4.4 96 31 98 8.8 95
    2X 5.0 110 31 100 9.2 100
    3X 5.2 114 27 85 9.1 99
    TFF-3 0 4.6 100 24 100 22 100
    (μg/mL) 1X 4.4 96 23 98 22 103
    2X 5.0 110 24 103 22 101
    3X 5.2 114 19 82 22 102
  • The results of the short-term stability assessment are summarized in Table 8. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any short-term storage.
  • TABLE 8
    Short-Term Stability of Analytes in Urine, Serum, and Plasma
    Storage Urine Sample Serum Sample Plasma Sample
    Time/ Sample Recovery Sample Recovery Sample Recovery
    Analyte Temp Conc. (%) Conc. (%) Conc. (%)
    Calbindin Control 226 100 33 100 7 100
    (ng/mL) 2 hr/ 242 107 30 90 6.3 90
    room
    temp
    2 hr. @ 228 101 29 89 6.5 93
    4° C.
    4 hr @ 240 106 28 84 5.6 79
    room
    temp
    4 hr. @ 202 89 29 86 5.5 79
    4° C.
    24 hr. @ 199 88 26 78 7.1 101
    4° C.
    Clusterin Control 185 100 224 100 171 100
    (ng/mL) 2 hr @ 173 94 237 106 180 105
    room
    temp
    2 hr. @ 146 79 225 100 171 100
    4° C.
    4 hr @ 166 89 214 96 160 94
    room
    temp
    4 hr. @ 157 85 198 88 143 84
    4° C.
    24 hr. @ 185 100 207 92 162 94
    4° C.
    CTGF Control 1.9 100 8.8 100 1.2 100
    (ng/mL) 2 hr @ 1.9 99 6.7 76 1 83
    room
    temp
    2 hr. @ 1.8 96 8.1 92 1.1 89
    4° C.
    4 hr @ 2.1 113 5.6 64 1 84
    room
    temp
    4 hr. @ 1.7 91 6.4 74 0.9 78
    4° C.
    24 hr. @ 2.2 116 5.9 68 1.1 89
    4° C.
    GST- Control 14 100 21 100 11 100
    alpha 2 hr @ 11 75 23 107 11 103
    (ng/mL) room
    temp
    2 hr. @ 13 93 22 104 9.4 90
    4° C.
    4 hr @ 11 79 21 100 11 109
    room
    temp
    4 hr. @ 12 89 21 98 11 100
    4° C.
    24 hr. @ 13 90 22 103 14 129
    4° C.
    KIM-1 Control 1.5 100 0.23 100 0.24 100
    (ng/mL) 2 hr @ 1.2 78 0.2 86 0.22 90
    room
    temp
    2 hr. @ 1.6 106 0.23 98 0.21 85
    4° C.
    4 hr @ 1.3 84 0.19 82 0.2 81
    room
    temp
    4 hr. @ 1.4 90 0.22 93 0.19 80
    4° C.
    24 hr. @ 1.1 76 0.18 76 0.23 94
    4° C.
    VEGF Control 851 100 1215 100 670 100
    (pg/mL) 2 hr @ 793 93 1055 87 622 93
    room
    temp
    2 hr. @ 700 82 1065 88 629 94
    4° C.
    4 hr @ 704 83 1007 83 566 84
    room
    temp
    4 hr. @ 618 73 1135 93 544 81
    4° C.
    24 hr. @ 653 77 1130 93 589 88
    4° C.
    β-2M Control 0.064 100 2.6 100 1.2 100
    (μg/mL) 2 hr @ 0.062 97 2.4 92 1.1 93
    room
    temp
    2 hr. @ 0.058 91 2.2 85 1.2 94
    4° C.
    4 hr @ 0.064 101 2.2 83 1.2 94
    room
    temp
    4 hr. @ 0.057 90 2.2 85 1.2 98
    4° C.
    24 hr. @ 0.06 94 2.5 97 1.3 103
    4° C.
    Cystatin C Control 52 100 819 100 476 100
    (ng/mL) 2 hr @ 50 96 837 102 466 98
    room
    temp
    2 hr. @ 44 84 884 108 547 115
    4° C.
    4 hr @ 49 93 829 101 498 105
    room
    temp
    4 hr. @ 46 88 883 108 513 108
    4° C.
    24 hr. @ 51 97 767 94 471 99
    4° C.
    NGAL Control 857 100 302 100 93 100
    (ng/mL) 2 hr @ 888 104 287 95 96 104
    room
    temp
    2 hr. @ 923 108 275 91 92 100
    4° C.
    4 hr @ 861 101 269 89 88 95
    room
    temp
    4 hr. @ 842 98 283 94 94 101
    4° C.
    24 hr. @ 960 112 245 81 88 95
    4° C.
    Osteopontin Control 2243 100 6.4 100 5.2 100
    (ng/mL) 2 hr @ 2240 100 6.8 107 5.9 114
    room
    temp
    2 hr. @ 2140 95 6.4 101 6.2 120
    4° C.
    4 hr @ 2227 99 6.9 108 5.8 111
    room
    temp
    4 hr. @ 2120 95 7.7 120 5.2 101
    4° C.
    24 hr. @ 2253 100 6.5 101 6 116
    4° C.
    TIMP-1 Control 17 100 349 100 72 100
    (ng/mL) 2 hr @ 17 98 311 89 70 98
    room
    temp
    2 hr. @ 16 94 311 89 68 95
    4° C.
    4 hr @ 17 97 306 88 68 95
    room
    temp
    4 hr. @ 16 93 329 94 74 103
    4° C.
    24 hr. @ 18 105 349 100 72 100
    4° C.
    A-1M Control 3.6 100 2.2 100 1 100
    (μg/mL) 2 hr @ 3.5 95 2 92 1 105
    room
    temp
    2 hr. @ 3.4 92 2.1 97 0.99 99
    4° C.
    4 hr @ 3.2 88 2.2 101 0.99 96
    room
    temp
    4 hr. @ 3 82 2.2 99 0.97 98
    4° C.
    24 hr. @ 3 83 2.2 100 1 101
    4° C.
    THP Control 1.2 100 34 100 2.1 100
    (μg/mL) 2 hr @ 1.2 99 34 99 2 99
    room
    temp
    2 hr. @ 1.1 90 34 100 2 98
    4° C.
    4 hr @ 1.1 88 27 80 2 99
    room
    temp
    4 hr. @ 0.95 79 33 97 2 95
    4° C.
    24 hr. @ 0.91 76 33 98 2.4 116
    4° C.
    TFF-3 Control 1230 100 188 100 2240 100
    (μg/mL) 2 hr @ 1215 99 179 95 2200 98
    room
    temp
    2 hr. @ 1200 98 195 104 2263 101
    4° C.
    4 hr @ 1160 94 224 119 2097 94
    room
    temp
    4 hr. @ 1020 83 199 106 2317 103
    4° C.
    24 hr. @ 1030 84 229 122 1940 87
    4° C.
  • The results of this experiment demonstrated that the analytes associated with renal disorders tested were suitably stable over several freeze/thaw cycles, and up to 24 hrs. of storage at a temperature of 4° C.
    • Example 8 Diagnosis of Renal Damage Using Detection of Analytes in Human Urine Samples
  • To assess the effectiveness of a human kidney toxicity panel to detect renal damage due to disease states, the following experiment was conducted. Urine samples were obtained from healthy control patients (n=5), renal cancer patients (n=4) and “other” cancer patients (n=8) afflicted with lung cancer, pancreatic cancer, liver cancer, or colon cancer. All urine samples were diluted as described in Example 4 and subjected to a sandwich-capture assay as described in Example 1. Urine concentrations of analytes included in a human kidney toxicity panel were measured by the assay, including alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.
  • FIG. 1 summarizes the urine concentrations of those analytes that differed significantly from control urine concentrations. The urine concentrations of A1M, NGAL, and THP were slightly elevated for the renal cancer patient group and more significantly elevated for the “other” cancer patient group. Urine B2M concentrations appeared to be elevated for both the renal cancer and “other” cancer patient groups, although the BRM concentrations exhibited more variability than the other analyte concentrations shown in FIG. 1.
  • The results of this experiment demonstrated that panels of analytes detected in urine samples were capable of identifying patients having renal damage resulting from renal cancer and other cancers.
    • Example 9 Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury
  • A screen for potential protein biomarkers in relation to kidney toxicity/damage was performed using a panel of biomarkers, in a set of urine and plasma samples from patients with documented renal damage. The investigated patient groups included diabetic nephropathy (DN), obstructive uropathy (OU), analgesic abuse (AA) and glomerulonephritis (GN) along with age, gender and BMI matched control groups. Multiplexed immunoassays were applied in order to quantify the following protein analytes: Alpha-1 Microglobulin (α1M), KIM-1, Microalbumin, Beta-2-Microglobulin (β32M), Calbindin, Clusterin, CystatinC, TreFoilFactor-3 (TFF-3), CTGF, GST-alpha, VEGF, Calbindin, Osteopontin, Tamm-HorsfallProtein (THP), TIMP-1 and NGAL.
  • Li-Heparin plasma and mid-stream spot urine samples were collected from four different patient groups. Samples were also collected from age, gender and BMI matched control subjects. 20 subjects were included in each group resulting in a total number of 160 urine and plasma samples. All samples were stored at −80° C. before use. Glomerular filtration rate for all samples was estimated using two different estimations (Modification of Diet in Renal Disease or MDRD, and the Chronic Kidney Disease Epidemiology Collaboration or CKD-EPI) to outline the eGFR (estimated glomerular filtration rate) distribution within each patient group (FIG. 2). Protein analytes were quantified in human plasma and urine using multiplexed immunoassays in the Luminex xMAP™ platform. The microsphere-based multiplex immunoassays consist of antigen-specific antibodies and optimized reagents in a capture-sandwich format. Output data was given as g/ml calculated from internal standard curves. Because urine creatinine (uCr) correlates with renal filtration rate, data analysis was performed without correction for uCr. Univariate and multivariate data analysis was performed comparing all case vs. control samples as well as cases vs. control samples for the various disease groups.
  • The majority of the measured proteins showed a correlation to eGFR. Measured variables were correlated to eGFR using Pearson's correlations coefficient, and samples from healthy controls and all disease groups were included in the analysis. 11 and 7 proteins displayed P-values below 0.05 for plasma and urine (Table 9) respectively.
  • TABLE 9
    Correlation analysis of eGFR and variables for all case samples
    URINE PLASMA
    Variable Pearson's r P-Value Variable Pearson's r  P-Value
    Alpha-1- −0.08 0.3 Alpha-1- −0.33
    Figure US20110065608A1-20110317-P00001
    Microglobulin Microglobulin
    Beta-2- −0.23 0.003 Beta-2- −0.39
    Figure US20110065608A1-20110317-P00001
    Microglobulin Microglobulin
    Calbindin −0.16 0.04 Calbindin −0.18 <0.02
    Clusterin −0.07 0.4 Clusterin −0.51
    Figure US20110065608A1-20110317-P00001
    CTGF −0.08 0.3 CTGF −0.05 0.5
    Creatinine −0.32
    Figure US20110065608A1-20110317-P00001
    		 Cystatin-C −0.42 <0.0001
    Cystatin-C −0.24 0.002 GST-alpha −0.12 0.1
    GST-alpha −0.11 0.2 KIM-1 −0.17 0.03
    KIM-1 −0.08 0.3 NGAL −0.28 <0.001
    Microalbumin_UR −0.17 0.03 Osteopontin −0.33
    Figure US20110065608A1-20110317-P00001
    NGAL −0.15 0.07 THP −0.31
    Figure US20110065608A1-20110317-P00001
    Osteopontin −0.19 0.02 TIMP-1 −0.28 <0.001
    THP −0.05 0.6 TFF3 −0.38
    Figure US20110065608A1-20110317-P00001
    TIMP-1 −0.19 0.01 VEGF −0.14 0.08
    TFF2 −0.09 0.3
    VEGF −0.07 0.4
    P values <0.0001 are shown in bold italics
    P values <0.005 are shown in bold
    P values <0.05 are shown in italics
  • For the various disease groups, univariate statistical analysis revealed that in a direct comparison (T-test) between cases and controls, a number of proteins were differentially expressed in both urine and plasma (Table 10 and FIG. 3). In particular, clusterin showed a marked differential pattern in plasma.
  • TABLE 10
    Differentially regulated proteins by
    univariate statistical analysis
    Group Matrix Protein p-value
    AA Urine Calbindin 0.016
    AA Urine NGAL 0.04
    AA Urine Osteopontin 0.005
    AA Urine Creatinine 0.001
    AA Plasma Calbindin 0.05
    AA Plasma Clusterin 0.003
    AA Plasma KIM-1 0.03
    AA Plasma THP 0.001
    AA Plasma TIMP-1 0.02
    DN Urine Creatinine 0.04
    DN Plasma Clusterin 0.006
    DN Plasma KIM-1 0.01
    GN Urine Creatinine 0.004
    GN Urine Microalbumin 0.0003
    GN Urine NGAL 0.05
    GN Urine Osteopontin 0.05
    GN Urine TFF3 0.03
    GN Plasma Alpha 1 Microglobulin 0.002
    GN Plasma Beta 2 Microglobulin 0.03
    GN Plasma Clusterin 0.00
    GN Plasma Cystatin C 0.01
    GN Plasma KIM-1 0.003
    GN Plasma NGAL 0.03
    GN Plasma THP 0.001
    GN Plasma TIMP-1 0.003
    GN Plasma TFF3 0.01
    GN Plasma VEGF 0.02
    OU Urine Clusterin 0.02
    OU Urine Microalbumin 0.007
    OU Plasma Clusterin 0.00
  • Application of multivariate analysis yielded statistical models that predicted disease from control samples (plasma results are shown in FIG. 4).
  • In conclusion, these results form a valuable base for further studies on these biomarkers in urine and plasma both regarding baseline levels in normal populations and regarding the differential expression of the analytes in various disease groups. Using this panel of analytes, error rates from adaboosting and/or random forest were low enough (<10%) to allow a prediction model to differentiate between control and disease patient samples. Several of the analytes showed a greater correlation to eGFR in plasma than in urine.
    • Example 10 Statistical Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury
  • Urine and plasma samples were taken from 80 normal control group subjects and 20 subjects from each of four disorders: analgesic abuse, diabetic nephropathy, glomerulonephritis, and obstructive uropathy. The samples were analyzed for the quantity and presence of 16 different proteins (alpha-1 microglobulin (α1M), beta-2 microglobulin (β2M), calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) as described in Example 1 above. The goal was to determine the analytes that distinguish between a normal sample and a diseased sample, a normal sample and an obstructive uropathy (OU) sample, and finally, an glomerulonephritis sample from the other disease samples (diabetic nephropathy (DN), analgesic abuse (AA), and glomerulonephritis (GN)).
  • From the above protein analysis data, bootstrap analysis was used to estimate the future performance of several classification algorithms. For each bootstrap run, training data and testing data was randomly generated. Then, the following algorithms were applied on the training data to generate models and then apply the models to the testing data to make predictions: automated Matthew's classification algorithm, classification and regression tree (CART), conditional inference tree, bagging, random forest, boosting, logistic regression, SVM, and Lasso. The accuracy rate and ROC areas were recorded for each method on the prediction of the testing data. The above was repeated 100 times. The mean and the standard deviation of the accuracy rates and of the ROC areas were calculated.
  • The mean error rates and AUROC were calculated from urine and AUROC was calculated from plasma for 100 runs of the above method for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 5, Table 11), AA vs. normal (FIG. 7, Table 13), DN vs. AA (FIG. 9, Table 15, AA vs. GN (FIG. 11, Table 17), and AA vs. OU (FIG. 13, Table 19).
  • The average relative importance of 16 different analytes (alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) and 4 different clinical variables (weight, BMI, age, and gender) from 100 runs were analyzed with two different statistical methods—random forest (plasma and urine samples) and boosting (urine samples)—for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 6, Table 12), AA vs. normal (FIG. 8, Table 14), DN vs. AA (FIG. 10, Table 16), AA vs. GN (FIG. 12, Table 18), and AA vs. OU (FIG. 14, Table 20).
  • TABLE 11
    Disease v. Normal
    Standard
    Mean deviation
    method AUROC AUROC
    random 0.931 0.039
    forest
    bagging 0.919 0.045
    svm 0.915 0.032
    boosting 0.911 0.06
    lasso 0.897 0.044
    logistic 0.891 0.041
    regression
    ctree 0.847 0.046
    cart 0.842 0.032
    matt 0.83 0.023
  • TABLE 12
    Disease v. Normal
    relative
    analyte importance
    Creatinine 11.606
    Kidney_Injury_M 8.486
    Tamm_Horsfall_P 8.191
    Total_Protein 6.928
    Osteopontin 6.798
    Neutrophil_Gela 6.784
    Tissue_Inhibito 6.765
    Vascular_Endoth 6.716
    Trefoil_Factor— 6.703
    Cystatin_C 6.482
    Alpha_1_Microgl 6.418
    Beta_2_Microglo 6.228
    Glutathione_S_T 6.053
    clusterin 5.842
  • TABLE 13
    AA v. NL
    Standard
    deviation
    Mean of
    method AUROC AUROC
    cart 1 0
    bagging 1 0
    boosting 1 0
    lasso 0.998 0.008
    ctree 0.998 0.015
    random 0.997 0.012
    forest
    svm 0.977 0.033
    logistic 0.933 0.092
    regression
    matt 0.873 0.112
  • TABLE 14
    AA v. NL
    Relative
    analyte importance
    Creatinine 17.800
    Tissue_Inhibito 9.953
    Total_Protein 8.837
    Tamm_Horsfall_P 7.379
    Cystatin_C 6.237
    Kidney_Injury_M 6.174
    Beta_2_Microglo 5.915
    Neutrophil_Gela 5.761
    Alpha_1_Microgl 5.742
    Trefoil_Factor— 5.736
    Osteopontin 5.561
    Vascular_Endoth 5.338
    clusterin 4.892
    Glutathione_S_T 4.675
  • TABLE 15
    AA v. DN
    Standard
    Mean deviation
    method AUROC AUROC
    lasso 0.999 0.008
    random 0.989 0.021
    forest
    svm 0.988 0.039
    boosting 0.988 0.022
    bagging 0.972 0.036
    logistic 0.969 0.057
    regression
    cart 0.93 0.055
    ctree 0.929 0.063
    matt 0.862 0.12
  • TABLE 16
    AA v. DN
    Relative
    analyte importance
    Creatinine 17.57
    Total_Protein 10.90
    Tissue_Inhibito 8.77
    clusterin 6.89
    Glutathione_S_T 6.24
    Alpha_1_Microgl 6.15
    Beta_2_Microglo 6.06
    Cystatin_C 5.99
    Trefoil_Factor— 5.88
    Kidney_Injury_M 5.49
    Vascular_Endoth 5.38
    Tamm_Horsfall_P 5.33
    Osteopontin 4.86
    Neutrophil_Gela 4.47
  • TABLE 17
    AA v. GN
    Standard
    deviation
    Mean of
    method AUROC AUROC
    svm 0.689 0.11
    boosting 0.675 0.102
    bagging 0.674 0.106
    random 0.66 0.096
    forest
    matt 0.631 0.085
    cart 0.626 0.089
    logistic 0.614 0.091
    regression
    lasso 0.606 0.102
    ctree 0.53 0.061
  • TABLE 18
    AA v. GN
    Relative
    analyte importance
    Creatinine 10.780
    Alpha_1_Microgl 8.847
    Kidney_Injury_M 8.604
    clusterin 8.109
    Total_Protein 7.679
    Glutathione_S_T 7.493
    Neutrophil_Gela 6.721
    Vascular_Endoth 6.461
    Cystatin_C 6.444
    Beta_2_Microglo 6.261
    Trefoil_Factor— 6.184
    Tamm_Horsfall_P 5.872
    Tissue_Inhibito 5.690
    Osteopontin 4.855
  • TABLE 19
    AA v. OU
    Standard
    deviation
    Mean of
    method AUROC AUROC
    random 0.814 0.11
    forest
    bagging 0.792 0.115
    svm 0.788 0.112
    lasso 0.786 0.118
    boosting 0.757 0.117
    matt 0.687 0.111
    logistic 0.683 0.116
    regression
    cart 0.665 0.097
    ctree 0.659 0.118
  • TABLE 20
    AA v. OU
    Relative
    analyte importance
    Total_Protein 11.502
    Tissue_Inhibito 9.736
    Cystatin_C 9.161
    Alpha_1_Microgl 8.637
    Trefoil_Factor— 7.329
    Osteopontin 7.326
    Beta_2_Microglo 6.978
    Neutrophil_Gela 6.577
    Glutathione_S_T 6.100
    Tamm_Horsfall_P 6.066
    Kidney_Injury_M 6.038
    Vascular_Endoth 5.946
    clusterin 4.751
    Creatinine 3.854
  • It should be appreciated by those of skill in the art that the techniques disclosed in the examples above represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Claims (36)

1. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
2. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol.
3. The assay device of claim 2, wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
4. The assay device of claim 2, wherein the renal disorder comprises obstructive uropathy, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, THP, A1M, clusterin, NGAL, and osteopontin.
5. The assay device of claim 2, wherein the renal disorder comprises obstructive uropathy, wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, THP, alpha-1 microglobulin, clusterin, NGAL, and osteopontin.
6. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
7. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.
8. The assay device of claim 2, wherein the renal disorder comprises kidney toxicity, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
9. The assay device of claim 2, wherein the renal disorder comprises kidney toxicity, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.
10. The assay device of claim 2, wherein the renal disorder comprises diabetic nephropathy, and wherein the three or more antibodies have antigenic determinants for analytes selected from the group consisting of microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
11. The assay device of claim 2, wherein the renal disorder comprises diabetic nephropathy, and wherein the panel of biomarkers has six antibodies having antigenic determinants for analytes selected from the group consisting of microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.
12. The assay device of claim 2, wherein the renal disorder comprises kidney transplant rejection and chronic allograft nephropathy, and wherein the panel comprises three or more antibodies having antigenic determinants for analytes selected from the group consisting of BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol.
13. The assay device of claim 2, wherein the panel of biomarkers comprises ten or more antibodies having antigenic determinants for analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
14. The assay device of claim 2, wherein the panel of biomarkers has sixteen antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
15. The assay device of claim 2, wherein the contact surface comprises a substrate capable of immobilizing analytes captured by the antibodies.
16. The assay device of claim 15, wherein the substrate comprises a porous material selected from the group consisting of paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, plastic strips, beads, the inner surface of a well, the surface of a microtitration tray, and combinations thereof.
17. The assay device of claim 2, further comprising a plurality of indicators, wherein one of the plurality of indicators is attached to one of the three or more antibodies
18. The assay device of claim 2, wherein the plurality of indicators comprises visual indicators and electrochemical indicators.
19. The assay device of claim 18, wherein the visual indicators are selected from the group consisting of nanoparticulate gold, polyurethane microspheres loaded with dye compounds, latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored product.
20. The assay device of claim 18, wherein the electrochemical indicators are selected from the group consisting of ascorbate, vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron, and mercury.
21. The assay device of claim 2, wherein the assay method comprises electrophoresis, mass spectrometry, protein microarrays, western blot, immunohistochemical staining, enzyme-linked immunosorbent assay methods, and particle-based capture-sandwich immunoassays.
22. The assay device of claim 2, wherein the renal disorder comprises glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, acute renal failure, chronic renal failure, nephrosis, nephropathy, polycystic kidney disease, Bright's disease, renal transplant, chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, and acute bilateral obstructive uropathy.
23. The assay device of claim 2, wherein the renal disorder comprises renal damage caused by exposure to secondary agents and conditions including therapeutic drugs, recreational drugs, contrast agents, toxins, nephrolithiasis, ischemia, liver transplantation, heart transplantation, lung transplantation, and hypovolemia.
24. The assay device of claim 2, wherein the renal disorder comprises renal damage secondary to a primary disease state including diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, and Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate diseases.
25. The assay device of claim 2, wherein the mammal is selected from the group consisting of humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen.
26. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising:
a. three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol;
b. three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies;
c. three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and
d. three or more indicators, wherein each of the indicators is attached to one of the detection antibodies.
27. The assay device of claim 26, wherein the three or more capture antibodies have antigenic determinants for the analytes selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
28. The assay device of claim 26, wherein the panel of biomarkers comprises six or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
29. The assay device of claim 26, wherein the panel of biomarkers comprises ten or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
30. The assay device of claim 26, wherein the panel of biomarkers comprises sixteen or more antibodies having antigenic determinants for the analytes comprising alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
31. A kit for diagnosing, monitoring, or determining a renal disorder in a mammal, the kit comprising:
a. the assay device of claim 2; and
b. a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
32. The kit of claim 31, wherein the collection apparatus comprises urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, and aspiration needles.
33. A kit for diagnosing, monitoring, or determining a renal disorder in a mammal, the kit comprising:
a. the assay device of claim 26; and
b. a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.
34. The kit of claim 33, wherein the collection apparatus comprises urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, and aspiration needles.
35. An assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
36. A platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.
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US14/643,873 Abandoned US20150276765A1 (en) 2009-08-07 2015-03-10 Methods and Devices for Detecting Diabetic Nephropathy and Associated Disorders
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US15/675,367 Abandoned US20170370947A1 (en) 2009-08-07 2017-08-11 Methods and Devices for Detecting Diabetic Nephropathy and Associated Disorders
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110174062A1 (en) * 2008-08-29 2011-07-21 Joseph Anderberg Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110195429A1 (en) * 2008-08-28 2011-08-11 Astute Medical Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110201038A1 (en) * 2008-10-21 2011-08-18 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
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US8486717B2 (en) 2011-01-18 2013-07-16 Symbolics, Llc Lateral flow assays using two dimensional features
WO2013158777A1 (en) * 2012-04-17 2013-10-24 Icahn School Of Medicine At Mount Sinai Method for predicting risk of exposure to interstitial fibrosis and tubular atrophy with clusterin
US8871459B2 (en) 2009-08-07 2014-10-28 Astute Medical, Inc. Method for evaluating renal status by determining beta-2-glycoprotein 1
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US9029093B2 (en) 2010-02-26 2015-05-12 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
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US9360488B2 (en) 2013-01-17 2016-06-07 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9414813B2 (en) 2009-02-16 2016-08-16 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US9599615B2 (en) 2013-03-13 2017-03-21 Symbolics, Llc Lateral flow assays using two dimensional test and control signal readout patterns
US9874556B2 (en) 2012-07-18 2018-01-23 Symbolics, Llc Lateral flow assays using two dimensional features
US10324093B2 (en) 2009-11-07 2019-06-18 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
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US10823742B2 (en) 2010-06-23 2020-11-03 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10830773B2 (en) 2009-12-20 2020-11-10 Astute Medical, Inc. Methods for prognosis of future acute renal injury and acute renal failure
US10928403B2 (en) 2010-06-23 2021-02-23 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10935548B2 (en) 2011-12-08 2021-03-02 Astute Medical, Inc. Methods for diagnosis and prognosis of renal injury and renal failure using insulin-like growth factor-binding protein 7 and metalloproteinase inhibitor 2
US11243217B2 (en) 2016-06-06 2022-02-08 Astute Medical, Inc. Management of acute kidney injury using insulin-like growth factor-binding protein 7 and tissue inhibitor of metalloproteinase 2
US11454635B2 (en) 2010-02-05 2022-09-27 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2767616A1 (en) 2009-07-09 2011-01-13 The Scripps Research Institute Gene expression profiles associated with chronic allograft nephropathy
WO2011085300A2 (en) * 2010-01-08 2011-07-14 The Penn State Research Foundation Compostions and methods relating to monitoring alcohol consumption and alcohol abuse
US20120094859A1 (en) * 2010-10-19 2012-04-19 Eva Redei Methods for detection of depressive disorders
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CA3133249C (en) 2012-02-09 2023-07-25 Memed Diagnostics Ltd. Signatures and determinants for diagnosing infections and methods of use thereof
FR2987244B1 (en) 2012-02-29 2015-09-04 Oreal APPLICATOR COMPRISING A ROD CONNECTED BY AN ARTICULATION TO AN APPLICATION ELEMENT
CN102749457A (en) * 2012-07-27 2012-10-24 北京恩济和生物科技有限公司 Beta2-microglobulin detection kit and preparing method thereof
JP6509119B2 (en) 2012-10-24 2019-05-08 インリージェン Renal cell population and use thereof
US9167240B1 (en) * 2012-12-12 2015-10-20 Board Of Regents Of The University Of Texas System Methods and compositions for validation of fluorescence imaging and tomography devices
US20160007900A1 (en) * 2013-03-01 2016-01-14 The Regents Of The University Of California Point-of-Care Device for Monitoring Renal Function
US9804154B2 (en) * 2013-03-12 2017-10-31 Epinex Diagnostics, Inc. Rapid test for urine albumin and urine creatinine
WO2015077676A1 (en) * 2013-11-23 2015-05-28 Singulex, Inc. Serum biomarkers in human kidney disease patients
US10451636B2 (en) 2014-04-09 2019-10-22 The Regents Of The University Of California Protein biomarkers for immune assessment and prediction of transplant rejection
US11104951B2 (en) 2014-05-22 2021-08-31 The Scripps Research Institute Molecular signatures for distinguishing liver transplant rejections or injuries
US10443100B2 (en) 2014-05-22 2019-10-15 The Scripps Research Institute Gene expression profiles associated with sub-clinical kidney transplant rejection
CN104198723A (en) * 2014-08-11 2014-12-10 南京普朗医疗设备有限公司 Rapid NGAL (Neutrophil Gelatinase Associated Lipocalin) detection kit based on amino acid spacer arm
CN111624346A (en) 2014-08-14 2020-09-04 米密德诊断学有限公司 Kit for predicting and/or determining prognosis
CN104215769A (en) * 2014-08-14 2014-12-17 上海睿康生物科技有限公司 Latex enhanced immunoturbidimetry NGAL detection kit
US11154712B2 (en) * 2014-08-28 2021-10-26 Medtronic Ardian Luxembourg S.A.R.L. Methods for assessing efficacy of renal neuromodulation and associated systems and devices
US20170315119A1 (en) * 2014-10-07 2017-11-02 University Health Network Urinary biomarkers for sle and lupus nephritis
US20170234873A1 (en) 2014-10-14 2017-08-17 Memed Diagnostics Ltd. Signatures and determinants for diagnosing infections in non-human subjects and methods of use thereof
KR102431003B1 (en) * 2014-10-20 2022-08-09 아스튜트 메디컬 인코포레이티드 Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20170269081A1 (en) * 2014-12-11 2017-09-21 Memed Diagnostics Ltd. Marker combinations for diagnosing infections and methods of use thereof
CN104833809A (en) * 2015-05-05 2015-08-12 南京闻智生物科技有限公司 Latex-enhanced immunonephelometry kit for determination of resistin, preparation method and detection method thereof
CL2015003047A1 (en) * 2015-10-15 2016-06-17 Univ Chile Ex vivo method to detect acute renal injury early in critically ill patients, which includes mediciom in a sample of three proteins as biomarkers, fibroblastic growth factor 23, klotho and erythropoietin
CN108699583B (en) 2016-03-03 2022-11-01 米密德诊断学有限公司 RNA determinants for distinguishing bacterial and viral infections
WO2018011795A1 (en) 2016-07-10 2018-01-18 Memed Diagnostics Ltd. Protein signatures for distinguishing between bacterial and viral infections
EP4184167A1 (en) 2016-07-10 2023-05-24 MeMed Diagnostics Ltd. Early diagnosis of infections
US11385241B2 (en) 2016-09-29 2022-07-12 Memed Diagnostics Ltd. Methods of prognosis and treatment
EP3519834A4 (en) 2016-09-29 2020-06-17 MeMed Diagnostics Ltd. Methods of risk assessment and disease classification
WO2018119626A1 (en) * 2016-12-27 2018-07-05 菲鹏生物股份有限公司 Assay kit for neutrophil gelatinase-associated lipocalin
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EP3568695A4 (en) 2017-01-12 2020-12-16 Astute Medical, Inc. Methods and compositions for evaluation and treatment of renal injury and renal failure based on c-c motif chemokine ligand 14 measurement
RU2677325C1 (en) * 2017-05-02 2019-01-16 Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт клинической и экспериментальной ревматологии" Method for diagnosis of renal damage in systemic lupus erythematosus
WO2018222865A1 (en) 2017-05-31 2018-12-06 Mars, Incorporated Methods of diagnosing and treating chronic kidney disease
US10209260B2 (en) 2017-07-05 2019-02-19 Memed Diagnostics Ltd. Signatures and determinants for diagnosing infections and methods of use thereof
CN107918020B (en) * 2017-11-15 2019-01-01 浙江夸克生物科技有限公司 Neutrophil gelatinase-associated lipocalin assay kit
JP7361698B2 (en) 2018-01-19 2023-10-16 マース インコーポレーテッド Biomarkers and classification algorithms for feline chronic kidney disease
US20210327589A1 (en) 2018-07-14 2021-10-21 Mars, Incorporated Biomarkers and test models for chronic kidney disease
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CN110412290A (en) * 2019-07-29 2019-11-05 冯仕品 SLE total disease mobility and kidney trouble mobility information detecting system
WO2021022167A2 (en) 2019-08-01 2021-02-04 The Research Institute At Nationwide Children's Hospital Compositions and methods for diagnosing urinary tract obstruction
US20210293719A1 (en) * 2020-03-23 2021-09-23 Johnson & Johnson Consumer Inc. System and method for measuring analyte concentration in bodily fluids
AU2021283177A1 (en) 2020-06-01 2022-12-08 Mars, Incorporated System and method for chronic kidney disease of a dog

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030199000A1 (en) * 2001-08-20 2003-10-23 Valkirs Gunars E. Diagnostic markers of stroke and cerebral injury and methods of use thereof
US20060008804A1 (en) * 2002-07-04 2006-01-12 Salah-Dine Chibout Marker genes
US20060269949A1 (en) * 2005-05-23 2006-11-30 Halloran Philip F Tissue rejection
US20070087448A1 (en) * 2004-02-16 2007-04-19 Nelsestuen Gary L Biological profiles and methods of use
US20070124086A1 (en) * 2001-05-22 2007-05-31 Mendrick Donna L Molecular nephrotoxicology modeling
US7235358B2 (en) * 2001-06-08 2007-06-26 Expression Diagnostics, Inc. Methods and compositions for diagnosing and monitoring transplant rejection
US20070248989A1 (en) * 2006-04-21 2007-10-25 Prasad Devarajan Method and Kit for the Early Detection of Impaired Renal Status
US20070287188A1 (en) * 2002-05-14 2007-12-13 Huaizhong Hu Systems and methods for identifying organ transplant risk
US20080087387A1 (en) * 2006-10-13 2008-04-17 Feng-Yuan Chen Blind bottom rail having a labor-saving function
US20080090304A1 (en) * 2006-10-13 2008-04-17 Barasch Jonathan Matthew Diagnosis and monitoring of chronic renal disease using ngal
US20080153092A1 (en) * 2006-09-05 2008-06-26 Stefan Kienle Markers of Renal Transplant Rejection and Renal Damage
US20080318803A1 (en) * 2004-05-27 2008-12-25 Vertex Pharmaceuticals Biomarkers for Monitoring Impdh Pathway Inhibition
US20090081713A1 (en) * 2007-09-20 2009-03-26 University Of Louisville Research Foundation, Inc. Peptide biomarkers predictive of renal function decline and kidney disease
US20090093010A1 (en) * 2004-09-21 2009-04-09 University Of Manitoba Method of Detecting Kidney Dysfunction
US20090176217A1 (en) * 2005-10-03 2009-07-09 Osnat Sella-Tavor Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis
US20090197287A1 (en) * 2002-12-06 2009-08-06 Renovar Incorporated Systems and methods for characterizing kidney disease

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004030521A2 (en) 2002-10-01 2004-04-15 The Johns Hopkins University Use of biomarkers for detecting acute renal transplant rejection
WO2005002416A2 (en) * 2003-06-04 2005-01-13 Joslin Diabetes Center, Inc. Predictors of renal disease
JP4741603B2 (en) 2004-12-20 2011-08-03 アンチボディショップ・アクティーゼルスカブ Measurement of neutrophil gelatinase-related lipocalin (NGAL) as a diagnostic marker for kidney injury
CA2596469A1 (en) * 2005-02-01 2006-08-10 Government Of The U.S.A, As Represented By The Secretary Department Of H Ealth & Human Services Biomarkers for tissue status
US20070087387A1 (en) 2005-04-21 2007-04-19 Prasad Devarajan Method for the Early Detection of Renal Disease Using Proteomics
JP5155857B2 (en) 2005-06-29 2013-03-06 モザイクヴェス ディアグノシュティクス アンド テラポイティクス アクチェン ゲゼルシャフト Polypeptide markers for early recognition of transplanted kidney rejection
US20070203216A1 (en) * 2006-02-14 2007-08-30 Bjarke Ebert Method of treating inflammatory diseases
US20070224643A1 (en) * 2006-03-09 2007-09-27 Mcpherson Paul H Methods and compositions for the diagnosis of diseases of the aorta
GB0607943D0 (en) * 2006-04-21 2006-05-31 Novartis Ag Biomarkers for chronic transplant dysfunction
WO2007138011A1 (en) 2006-05-25 2007-12-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for diagnosing and treating graft rejection and inflammatory conditiions
WO2008005375A2 (en) * 2006-06-30 2008-01-10 Merck & Co., Inc. Kidney toxicity biomarkers
US20100197510A1 (en) 2007-03-08 2010-08-05 Michael Spain Methods for rapid disease screening
EP2479571A3 (en) 2007-03-26 2012-09-26 Novartis AG Predictive renal safety biomarkers and biomarker signatures to monitor kidney function
WO2008128043A2 (en) 2007-04-11 2008-10-23 The General Hospital Corporation Diagnostic and prognostic methods for renal cell carcinoma
US20100184110A1 (en) * 2007-06-06 2010-07-22 Siemens Healthcare Diagnostics Inc. Predictive diagnostics for kidney disease
DE102008022609B4 (en) * 2008-05-05 2011-07-28 Otto-von-Guericke-Universität Magdeburg Medizinische Fakultät, 39120 Method for detecting the presence of kidney stones and / or inflammation of the urinary tract
US8394639B2 (en) * 2008-07-17 2013-03-12 Georg-August-Universitat Gottingen Stiftung Offentlichen Rechts, Universitatsmedizin Biomarkers for renal disease
WO2010022210A2 (en) 2008-08-21 2010-02-25 Pxbiosciences Llc Diagnosis and monitoring of renal failure using peptide biomarkers
US20100125288A1 (en) * 2008-11-17 2010-05-20 G&L Consulting, Llc Method and apparatus for reducing renal blood pressure

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070124086A1 (en) * 2001-05-22 2007-05-31 Mendrick Donna L Molecular nephrotoxicology modeling
US7235358B2 (en) * 2001-06-08 2007-06-26 Expression Diagnostics, Inc. Methods and compositions for diagnosing and monitoring transplant rejection
US20030199000A1 (en) * 2001-08-20 2003-10-23 Valkirs Gunars E. Diagnostic markers of stroke and cerebral injury and methods of use thereof
US20070287188A1 (en) * 2002-05-14 2007-12-13 Huaizhong Hu Systems and methods for identifying organ transplant risk
US20060008804A1 (en) * 2002-07-04 2006-01-12 Salah-Dine Chibout Marker genes
US20090197287A1 (en) * 2002-12-06 2009-08-06 Renovar Incorporated Systems and methods for characterizing kidney disease
US20070087448A1 (en) * 2004-02-16 2007-04-19 Nelsestuen Gary L Biological profiles and methods of use
US20080318803A1 (en) * 2004-05-27 2008-12-25 Vertex Pharmaceuticals Biomarkers for Monitoring Impdh Pathway Inhibition
US20090093010A1 (en) * 2004-09-21 2009-04-09 University Of Manitoba Method of Detecting Kidney Dysfunction
US20060269949A1 (en) * 2005-05-23 2006-11-30 Halloran Philip F Tissue rejection
US20090176217A1 (en) * 2005-10-03 2009-07-09 Osnat Sella-Tavor Novel nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis
US20070248989A1 (en) * 2006-04-21 2007-10-25 Prasad Devarajan Method and Kit for the Early Detection of Impaired Renal Status
US20080153092A1 (en) * 2006-09-05 2008-06-26 Stefan Kienle Markers of Renal Transplant Rejection and Renal Damage
US20080087387A1 (en) * 2006-10-13 2008-04-17 Feng-Yuan Chen Blind bottom rail having a labor-saving function
US20080090304A1 (en) * 2006-10-13 2008-04-17 Barasch Jonathan Matthew Diagnosis and monitoring of chronic renal disease using ngal
US20090081713A1 (en) * 2007-09-20 2009-03-26 University Of Louisville Research Foundation, Inc. Peptide biomarkers predictive of renal function decline and kidney disease

Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11150250B2 (en) 2008-08-28 2021-10-19 Astute Medical, Inc. Methods for diagnosing acute kidney injury or renal failure
US20110195429A1 (en) * 2008-08-28 2011-08-11 Astute Medical Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9057735B2 (en) 2008-08-29 2015-06-16 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110174062A1 (en) * 2008-08-29 2011-07-21 Joseph Anderberg Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US8778615B2 (en) 2008-10-21 2014-07-15 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US11754566B2 (en) 2008-10-21 2023-09-12 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110207161A1 (en) * 2008-10-21 2011-08-25 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10823733B2 (en) 2008-10-21 2020-11-03 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110201038A1 (en) * 2008-10-21 2011-08-18 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US8993250B2 (en) 2008-11-10 2015-03-31 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US20110229915A1 (en) * 2008-11-22 2011-09-22 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9229010B2 (en) 2009-02-06 2016-01-05 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10076314B2 (en) 2009-02-16 2018-09-18 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US9414813B2 (en) 2009-02-16 2016-08-16 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US9462998B2 (en) 2009-02-16 2016-10-11 Express Diagnostics Int'l, Inc. Device for assaying analytes in bodily fluids
US8871459B2 (en) 2009-08-07 2014-10-28 Astute Medical, Inc. Method for evaluating renal status by determining beta-2-glycoprotein 1
US10324093B2 (en) 2009-11-07 2019-06-18 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10830773B2 (en) 2009-12-20 2020-11-10 Astute Medical, Inc. Methods for prognosis of future acute renal injury and acute renal failure
US11262363B2 (en) 2009-12-20 2022-03-01 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US11454635B2 (en) 2010-02-05 2022-09-27 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9029093B2 (en) 2010-02-26 2015-05-12 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10928403B2 (en) 2010-06-23 2021-02-23 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US11761967B2 (en) 2010-06-23 2023-09-19 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US10823742B2 (en) 2010-06-23 2020-11-03 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
WO2012094657A1 (en) * 2011-01-08 2012-07-12 Astute Medical, Inc. Method and compositions for diagnosis and prognosis of renal injury and renal failure
US9851366B2 (en) 2011-01-18 2017-12-26 Symbolics, Llc Lateral flow assays using two dimensional features
US9874576B2 (en) 2011-01-18 2018-01-23 Symbolics, Llc Lateral flow assays using two dimensional features
US8486717B2 (en) 2011-01-18 2013-07-16 Symbolics, Llc Lateral flow assays using two dimensional features
US11016090B2 (en) 2011-01-18 2021-05-25 Symbolics, Llc Lateral flow assays using two dimensional features
US10935548B2 (en) 2011-12-08 2021-03-02 Astute Medical, Inc. Methods for diagnosis and prognosis of renal injury and renal failure using insulin-like growth factor-binding protein 7 and metalloproteinase inhibitor 2
WO2013158777A1 (en) * 2012-04-17 2013-10-24 Icahn School Of Medicine At Mount Sinai Method for predicting risk of exposure to interstitial fibrosis and tubular atrophy with clusterin
US10725026B2 (en) 2012-04-17 2020-07-28 Icahn School Of Medicine At Mount Sinai Method for predicting risk of exposure to interstitial fibrosis and tubular atrophy with clusterin
US9816985B2 (en) 2012-04-17 2017-11-14 Icahn School Of Medicine At Mount Sinai Method for predicting risk of exposure to interstitial fibrosis and tubular atrophy with clusterin
US9874556B2 (en) 2012-07-18 2018-01-23 Symbolics, Llc Lateral flow assays using two dimensional features
US9696322B2 (en) 2013-01-17 2017-07-04 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US11099194B2 (en) 2013-01-17 2021-08-24 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9360488B2 (en) 2013-01-17 2016-06-07 Astute Medical, Inc. Methods and compositions for diagnosis and prognosis of renal injury and renal failure
US9599615B2 (en) 2013-03-13 2017-03-21 Symbolics, Llc Lateral flow assays using two dimensional test and control signal readout patterns
US11243217B2 (en) 2016-06-06 2022-02-08 Astute Medical, Inc. Management of acute kidney injury using insulin-like growth factor-binding protein 7 and tissue inhibitor of metalloproteinase 2
WO2020072533A1 (en) * 2018-10-05 2020-04-09 The Regents Of The University Of California Kidney health monitoring in hypertension patients
EP3861353A4 (en) * 2018-10-05 2022-09-07 The Regents of the University of California Kidney health monitoring in hypertension patients
KR20200083344A (en) * 2018-12-28 2020-07-08 서울대학교산학협력단 Composition for diagnosing polycystic kidney disease comprising agent for detecting CTGF and use thereof
KR102288447B1 (en) * 2018-12-28 2021-08-10 서울대학교산학협력단 Composition for diagnosing polycystic kidney disease comprising agent for detecting CTGF and use thereof

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EP2462438A1 (en) 2012-06-13
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EP2462437A4 (en) 2013-01-30
US8735080B2 (en) 2014-05-27
WO2011017685A1 (en) 2011-02-10
US20210223267A1 (en) 2021-07-22
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EP2462445A1 (en) 2012-06-13
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US20170370947A1 (en) 2017-12-28
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