WO2008092214A1

WO2008092214A1 - Biomarkers for diabetes

Info

Publication number: WO2008092214A1
Application number: PCT/AU2008/000128
Authority: WO
Inventors: Agnes Cecile Debril; Bradley John Walsh; Geoffrey Bruce Randall
Original assignee: Minomic International Limited
Priority date: 2007-02-02
Filing date: 2008-02-04
Publication date: 2008-08-07

Abstract

The present invention relates to an assay for testing a subject for diabetes or a predisposition to diabetes comprising analysing a biological fluid from a subject for the presence of one or more biomarkers selected from Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1- antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1- microglobulin [Precursor] proteins or respective isoforms, peptides or fragments thereof, wherein detection of the biomarker is indicative of diabetes or a predisposition to diabetes in the subject.

Description

BIOMARKERS FOR DIABETES

Technical Field

The invention relates to biomarkers in the form of proteins and peptides for diabetes.

Background Art

Type Il diabetes is a metabolic disease characterized by hyperglycemia. In healthy people, when there is too much glucose in the blood, the glucose stimulates the pancreas to produce insulin. Insulin targets are fat, muscle cells and fat cells. Insulin binds to insulin receptor on the cells and activates cascade pathways leading to the entrance of glucose into the cells. Moreover, insulin also binds receptors in the liver and stops the liver from producing glucose.

In Type Il diabetic people, either the body doesn't produce enough insulin or the cells ignore insulin. When the glucose binds to the cell membrane, the different cascades do not proceed. Therefore, instead of being absorbed by the cells, the glucose remains in blood leading to hyperglycemia.

Type Il diabetes is a worldwide major public healthcare problem. It affects 194 million people in the world according to the latest World Health Organisation estimate (2000). With 17 million people suffering from diabetes, the United States of America is the most affected country. Australia counted 430,000 diabetic people in 2000. Among diabetic people, 35 % remain undiagnosed. Moreover, diabetes is one of the leading causes of death and disability in the US. Diabetes can lead to renal disease (nephropathy), micro vascular problems, blindness (retinopathy), extremity amputation and hypertension. Diabetes is projected to become one of the world's main disablers and killers within the next twenty-five years. In 2025, the figure of diabetic people worldwide is expected to increase to almost 300 million. According to the American Diabetes Association 2002, the global cost of healthcare for diabetes in the USA was estimated at $132 billion, with direct medical costs reaching $92 billion and indirect costs for disability, work loss and premature mortality costing $40 billion.

In order to identify proteins, western blotting and other immunological methods are the most successful techniques previously employed. However, these techniques are limited by the availability of specific antibodies and by the ability to examine only few proteins in each experiment.

Proteomics has recently emerged and has been developed for the large scale study of protein patterns in organisms. Typical goals for proteomic analysis are identification and quantification of proteins present in a specific tissue under specific circumstances. Proteomic technologies, in combination with bioinformatics, are powerful tools for proteins identification and characterisation. Commonly, two dimensional (2D) electrophoresis is used for proteins separation and Mass Spectrometry followed by databank searching are used for protein identification. Up to 10000 proteins can be studied simultaneously.

Diagnostics are a major application of proteomics fields, characterised by the search of biomarkers in body fluids, such as plasma or urine. Most commonly, the excretion of urine serves for flushing waste molecules collected from the blood by the kidneys, and for the homeostasis of the body liquids. Urine has a number of advantages as a fluid for biomarker investigation. First, urine can be obtained relatively easily from patients by non-invasive methods. Moreover, urine contains a lot of proteins and as a specific filtrate of blood, the proteins present in the urine are qualitatively the same as in the blood. Approximately 30% of urinary proteins are plasma proteins, whereas the other 70% are produced in the kidney. Finally, peptides have the ability to go through compartments in the organism. Therefore, many pathogenic problems can be reflected by changes in the protein composition in urine.

In the last few years, there has been an increasing interest in exploring the human urinary proteome. In particular, efforts have focused on developing strategies to generate reproducible protein maps of normal urine in order to compare protein expression between different groups. Heine and his colleagues have identified 34 peptides and proteins fragments by coupling HPLC and Electrospray Mass Spectrometry (Heine et a/., 1997). However, the identified peptides and proteins were limited to major abundant proteins. More recently, 124 gene products (proteins and EST's) were identified using liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS) (Spahr et al., 2001 ).

While many urinary proteins were identified by gel-free proteomic technologies, other research has been performed to identify urinary proteins in gel-based analyses using 2D electrophoresis to separate proteins. A 2D proteome map for normal human urinary proteins isolated by acetone precipitation and ultracentrifugation has been built (Thongboonkerd et al., 2002). Using MALDl, a total of 67 protein isoforms of 47 unique proteins were identified.

The present applicant previously performed proteomic analysis on diabetic and control urine samples using LC-MS tools. Seven glycosylated proteins were found in diabetic urine: alpha-2-macroglobulin, Apolipoprotein A1 , Immunoglobulin beta-heavy chain constant region, Chain A of Human IgAI , Inter-alpha-trypsin inhibitor heavy chain H4 precursor and Apolipoprotein B-100 (WO 2005/024429). After validation, those proteins could be used as potential biomarkers for diagnosis purposes in order to screen the population. By the use of 2D electrophoresis on control and diabetic urine, followed by image analysis, further potential biomarkers for Type Il diabetes have been identified by the present inventors.

Disclosure of Invention The present inventors have identified a number of protein biomarkers for Type Il diabetes. Eight proteins, and their isoforms were selected. Some of the biomarkers are isoforms of each other and differ only in their post- translational modifications. The preferred biomarkers are typically defined by their Swiss Prot accession numbers as most proteins end up with two to three names when they are "discovered" and named by different groups. Thus the designations TRFEJHUMAN, KNG1_HUMAN,

PIGRJ-IUMAN, A1ATJHUMAN, CD14_HUMAN, A2GLJHUMAN, DNAS1JHUMAN and AMBP_HUMAN define these proteins.

It will be appreciated that as the identified biomarkers are protein precursors, any end protein product and isoforms are covered by the present invention. Their most common names, designation and swissprot accession number as listed above are:

Serotransferrin [Precursor] (SEQ ID NO 1)

MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQS FRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVT LDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVV KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPE PRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCST LNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRD QYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKED LIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKP VKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKI MNGEADAMSL DGGFVYIAGK CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWN IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLAR APNHAWTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCL FRSETKDLLF RDDTVCLAKL HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP

^■5

Kininogen [Precursor] (SEQ ID NO 2)

MKLITILFLC SRLLLSLTQE SQSEEIDCND KDLFKAVDAA LKKYNSQNQS NNQFVLYRIT EATKTVGSDT FYSFKYEIKE GDCPVQSGKT WQDCEYKDAA KAATGECTAT VGKRSSTKFS VATQTCQITP AEGPWTAQY DCLGCVHPIS TQSPDLEPIL RHGIQYFNNN TQHSSLFMLN 0 EVKRAQRQW AGLNFRITYS IVQTNCSKEN FLFLTPDCKS LWNGDTGECT DNAYIDIQLR lASFSQNCDI YPGKDFVQPP TKICVGCPRD IPTNSPELEE TLTHTITKLN AENNATFYFK IDNVKKARVQ WAGKKYFID FVARETTCSK ESNEELTESC ETKKLGQSLD CNAEVYWPW EKKIYPTVNC QPLGMISLMK RPPGFSPFRS SRIGEIKEET TVSPPHTSMA PAQDEERDSG

KEQGHTRRHD WGHEKQRKHN LGHGHKHERD QGHGHQRGHG LGHGHEQQHG 5 LGHGHKFKLD DDLEHQGGHV LDHGHKHKHG HGHGKHKNKG KKNGKHNGWK

TEHLASSSED STTPSAQTQE KTEGPTPIPS LAKPGVTVTF SDFQDSDLIA TMMPPISPAP IQSDDDWIPD IQIDPNGLSF NPISDFPDTT SPKCPGRPWK SVSEINPTTQ MKESYYFDLT DGLS 0 . Polymeric-lmmunoglobulin Receptor [Precursor] (SEQ ID NO 3)

MLLFVLTCLL AVFPAISTKS PIFGPEEVNS VEGNSVSITC YYPPTSVNRH TRKYWCRQGA RGGCITLISS EGYVSSKYAG RANLTNFPEN GTFWNIAQL SQDDSGRYKC GLGINSRGLS FDVSLEVSQG PGLLNDTKVY TVDLGRTVTI NCPFKTENAQ KRKSLYKQIG LYPVLVIDSS GYVNPNYTGR IRLDIQGTGQ LLFSWINQL RLSDAGQYLC QAGDDSNSNK KNADLQVLKP5 EPELVYEDLR GSVTFHCALG PEVANVAKFL CRQSSGENCD VWNTLGKRA PAFEGRILLN PQDKDGSFSV VITGLRKEDA GRYLCGAHSD GQLQEGSPIQ AWQLFVNEES TIPRSPTWK GVAGSSVAVL CPYNRKESKS IKYWCLWEGA QNGRCPLLVD SEGWVKAQYE GRLSLLEEPG NGTFTVILNQ LTSRDAGFYW CLTNGDTLWR TTVEIKIIEG EPNLKVPGNV TAVLGETLKV PCHFPCKFSS YEKYWCKWNN TGCQALPSQD EGPSKAFVNC DENSRLVSLT LNLVTRADEG0 WYWCGVKQGH FYGETAAVYV AVEERKAAGS RDVSLAKADA APDEKVLDSG FREIENKAIQ DPRLFAEEKA VADTRDQADG SRASVDSGSS EEQGGSSRAL VSTLVPLGLV LAVGAVAVGV ARARHRKNVD RVSIRSYRTD ISMSDFENSR EFGANDNMGA SSITQETSLG GKEEFVATTE STTETKEPKK AKRSSKEEAE MAYKDFLLQS STVAAEAQDG PQEA 5 Alpha-1 -antitrypsin [Precursor] (SEQ ID NO 4)

MPSSVSWGIL LLAGLCCLVP VSLAEDPQGD AAQKTDTSHH DQDHPTFNKI TPNLAEFAFS LYRQLAHQSN STNIFFSPVS IATAFAMLSL GTKADTHDEI LEGLNFNLTE IPEAQIHEGF

QELLRTLNQP DSQLQLTTGN GLFLSEGLKL VDKFLEDVKK LYHSEAFTVN FGDTEEAKKQ

INDYVEKGTQ GKIVDLVKEL DRDTVFALVN YIFFKGKWER PFEVKDTEEE DFHVDQVTTV KVPMMKRLGM FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENE LTHDIiTKFL ENEDRRSASL HLPKLSITGT YDLKSVLGQL GITKVFSNGA DLSGVTEEAP LKLSKAVHKA VLTIDEKGTE AAGAMFLEAI PMSIPPEVKF NKPFVFLMIE QNTKSPLFMG KVVNPTQK

Monocyte differenciation antigen CD14 [Precursor] (SEQ ID NO 5)

MERASCLLLL LLPLVHVSAT TPEPCELDDE DFRCVCNFSE PQPDWSEAFQ CVSAVEVEIH AGGLNLEPFL KRVDADADPR QYADTVKALR VRRLTVGAAQ VPAQLLVGAL RVLAYSRLKE LTLEDLKITG TMPPLPLEAT GLALSSLRLR NVSWATGRSW LAELQQWLKP GLKVLSIAQA HSPAFSCEQV RAFPALTSLD LSDNPGLGER GLMAALCPHK FPAIQNLALR NTGMETPTGV CAALAAAGVQ PHSLDLSHNS LRATVNPSAP RCMWSSALNS LNLSFAGLEQ VPKGLPAKLR VLDLSCNRLN RAPQPDELPE VDNLTLDGNP FLVPGTALPH EGSMNSGVVP ACARSTLSVG

VSGTLVLLQG ARGFA

Leucine-rich alpha-2-glycoprotein [Precursor] (SEQ ID NO 6) MSSWSRQRPK SPGGIQPHVS RTLFLLLLLA ASAWGVTLSP KDCQVFRSDH GSSISCQPPA EIPGYLPADT VHLAVEFFNL THLPANLLQG ASKLQELHLS SNGLESLSPE FLRPVPQLRV LDLTRNALTG LPPGLFQASA TLDTLVLKEN QLEVLEVSWL HGLKALGHLD LSGNRLRKLP

PGLLANFTLL RTLDLGENQL ETLPPDLLRG PLQ'LERLHLE GNKLQVLGKD LLLPQPDLRY

LFLNGNKLAR VAAGAFQGLR QLDMLDLSNN SLASVPEGLW ASLGQPNWDM RDGFDISGNP WICDQNLSDL YRWLQAQKDK MFSQNDTRCA GPEAVKGQTL LAVAKSQ

Deoxyribonuclease 1 [Precursor] (SEQ ID NO 7)

MRGMKLLGAL LALAALLQGA VSLKIAAFNI QTFGETKMSN ATLVSYIVQI LSRYDIALVQ EVRDSHLTAV GKLLDNLNQD APDTYHYVVS EPLGRNSYKE RYLFVYRPDQ VSAVDSYYYD DGCEPCGNDT FNREPAIVRF FSRFTEVREF AIVPLHAAPG DAVAEIDALY DVYLDVQEKW GLEDVMLMGD FNAGCSYVRP SQWSSIRLWT SPTFQWLIPD SADTTATPTH CAYDRIVVAG MLLRGAVVPD SALPFNFQAA YGLSDQLAQA ISDHYPVEVM LK

Alpha-1 -microglobulin [Precursor] (SEQ ID NO 8) MRSLGALLLL LSACLAVSAG PVPTPPDNIQ VQENFNISRI YGKWYNLAIG STCPWLKKIM

DRMTVSTLVL GEGATEAEIS MTSTRWRKGV CEETSGAYEK TDTDGKFLYH KSKWNITMES YVVHTNYDEY AIFLTKKFSR HHGPTITAKL YGRAPQLRET LLQDFRVVAQ GVGIPEDSIF TMADRGECVP GEQEPEPILI PRVRRAVLPQ EEEGSGGGQL VTEVTKKEDS CQLGYSAGPC MGMTSRYFYN GTSMACETFQ YGGCMGNGNN FVTEKECLQT CRTVAACNLP IVRGPCRAFI QLWAFDAVKG KCVLFPYGGC QGNGNKFYSE KECREYCGVP GDGDEELLRF SN

In a first aspect, the present invention provides an assay for testing a subject for diabetes or a predisposition to diabetes comprising: analysing a biological fluid from a subject for the presence of one or more biomarkers selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or respective isoforms, peptides or fragments thereof, wherein detection of the biomarker is indicative of diabetes or a predisposition to diabetes in the subject.

Preferably, the diabetes is Type Il diabetes.

It will be appreciated that the biomarkers found by the present inventors as being indicative of diabetes or a predisposition to diabetes may be identified by detecting the whole protein or fragments thereof in a biological fluid.

The biological fluid can be any suitable fluid such as urine, saliva, blood, blood products such as serum, plasma, tears, cerebrospinal fluid, and lymph. The biological fluid can be assayed neat or concentrated or fractionated prior to assaying.

Preferably, the biological fluid is urine. In one preferred form, proteins present in the biological sample are digested to form peptide fragments which are detected by conducting mass spectrophotometric analysis on the sample in a manner effective to maximize elucidation of discernible peptide fragments contained therein; and comparing mass spectrum profiles of peptides forming Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective isoforms to mass spectrum profiles of peptides elucidated from the sample; wherein recognition of a mass spectrum profile in the sample displaying the mass spectrum profile for any one or more of proteins or peptides having amino acid selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or isoforms thereof is indicative of diabetes or a predisposition to diabetes.

In another preferred form, an antibody which recognises a protein selected from Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective isoforms or peptides or fragments thereof is used to probe or analyse the sample for the presence of one or more of the proteins.

The present inventors have found that urine from Type Il diabetic subjects or control subjects can have detectable levels of one or more biomarkers selected from Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective isoforms or peptides or fragments thereof. Preferably, the subject is a human.

The biomarkers according to the invention were obtained by: concentrating / desalting urine samples from diabetics or healthy individuals; separating proteins present in the concentrated urine samples; and identifying protein, protein fragment or peptide present in larger amounts or only detectable in the urine of diabetics or healthy individuals, but not both.

The urine is preferably concentrated / desalted by trichloroacetic acid precipitation. Alternatively urine can be concentrated by precipitation using acetone or membrane-based separation. It will be appreciated that other forms of concentration known to the art could also be used in this regard. The peptides or proteins can be separated by chromatography and identified by mass spectrometry.

It will be appreciated that when biomarkers have been identified, detection by any suitable means of their presence in a biological sample can be used as a diagnostic or screen. Preferably, the protein fragment may be as small as an antigenic peptide having about 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more amino acids.

In a second aspect, the present invention provides one or more isolated antibodies directed to a protein, protein fragment or peptide detectable in a biological sample of a subject being indicative of diabetes or a predisposition to diabetes in a subject, wherein the protein, protein fragment or peptide is selected from SEQ ID NO:1 , SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or isoforms thereof. In one preferred form, the antibody is a polyclonal antibody, which is derived by immunising mice or other suitable animal with one or more proteins, protein fragments or peptides found to be suitable biomarkers for diabetes.

In another preferred form, the antibody is an isolated monoclonal antibody to one or more proteins, protein fragments or peptides found to be suitable biomarkers for diabetes. Methods for developing monoclonal antibodies are well known to the art.

In another preferred form, a combination of several isolated monoclonal and/or polyclonal antibodies to one or more proteins, protein fragments or peptides found to be suitable biomarkers for diabetes is used. It will be appreciated that when an animal has raised an immune response to one or more biomarkers according to the present invention, hyperimmune serum or ascites fluid, for example, can be collected by usual methods. Specific antibodies can be obtained by separation methods known to the art such as precipitation, affinity chromatography, Protein A separation. The separated sera or ascites fluid can be used whole, diluted or as a starting material for separation of one or more peptides according to the first aspect of the present invention.

In one preferred form, the antibodies are detectably labelled. In one preferred form, the label is fluorochrome fluoresein isothiocyanate (FITC). Other labels such as Texas Red, Oregon Green, TRITC, Alexa dyes, allophycocyanin or rhodamine would also be suitable for the present invention. In another preferred form, the antibodies are radioactively labelled.

The assay may be an ELISA assay or radioimmunoassay. Other suitable assays utilizing antibodies are well known to the art and include protein chip based matrices.

In a third aspect, the present invention provides an assay for testing a subject for diabetes or a predisposition to diabetes comprising: obtaining a sample from a subject; and analysing the sample for the presence or amount of one or more biomarkers selected from Serotransferrin [Precursor], Kininogen [Precursor], Polymeric- lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective^* isoforms or peptides or fragments thereof, wherein the presence or amount of a biomarker is indicative of diabetes or a predisposition to diabetes in the subject.

The amount of biomarker can be defined as absolute, or normalized by the creatinine concentration of the sample. The assay may further include: concentrating the sample, and/or clean-up processing such as but not restricted to centrifugal filtration and solid phase extraction.

The assay may further include: measuring the creatinine concentration of the sample; digesting proteins present in the sample to form peptides; and optionally, separating the peptides. The sample can be any suitable sample. Preferably the sample is urine.

Preferably, the one or more proteins, protein fragments or peptides are detected by the use of an antibody according to second aspect of the present invention. In a fourth aspect, the' present invention provides a kit for assaying a subject for diabetes or a predisposition to diabetes comprising: one or more antibodies according to the second aspect of the present invention; and suitable reagents and diluents for the assay. In a fifth aspect, the present invention provides use of a biomarker selected from

Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective isoforms or peptides or fragments thereof in an assay for diabetes or a predisposition to diabetes.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this specification. In order that the present invention may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

Figure 1 shows 2D gel electrophoresis of type Il diabetes urine samples desalted and concentrated by 20% TCA precipitation, pH 4-7, Criterion 8-16%, SYPRO staining.

Figure 2 shows 2D gel electrophoresis of control urine samples desalted and concentrated by 20% TCA precipitation, pH 4-7, Criterion 8-16%, SYPRO staining.

Figure 3 shows Spot detection on reference gel using TT900 alignment sofware and Progenesis Software (PG 240 version 2006, Nonϋnear). Figure 4 shows the average control summary map with selected biomarkers and identifications: the bold outlines represent spots that are up-regulated in controls, and the italics outlines represent spots that are up-regulated on type Il diabetes. Post- translational modifications among selected biomarkers are emphasized by circles.

Figure 5 shows the standard curves obtained for Kininogen ELISA assay (top) and Transferrin ELISA assay (bottom), allowing the measurement of Kininogen and Transferrin concentrations in urine.

Mode(s) for Carrying Out the Invention

It has now been determined and is the subject of the present invention that protein biomarkers for Type Il diabetes are present in urine. 2D electrophoresis was performed on control and diabetic urine, followed by image analysis, in order to select spots that could contain potential biomarkers. The selected spots were cut, digested and analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in order to identify the potential biomarkers. Two ELISA assays have been developed and optimised using polyclonal and monoclonal antibodies to measure the amount of Serotransferrin and High Molecular Weight Kininogen in urine. MATERIALS AND METHODS Sample collection and storage

Fifty millilitres of morning urine were collected from Type Il diabetic patients and healthy controls. Samples were centrifuged for 15 min at 2,50Og in order to remove cellular residues. Supernatant was stored at -8O⁰C in order to inhibit protease activity, protein modification and microbiological contamination.

Sample semi-quantitation SDS-Page of urine sample were undertaken in order to evaluate relative protein concentration between samples. 20 ul sample was diluted 1:1 in SDS-PAGE boiling buffer (63 mM Tris-HCI, 10% Glycerol, 2% SDS, 25 mM DTT, Bromophenol Blue dye). After heating sample at 95⁰C for 5 min prior to loading on either Tris-Hepes Long Life gels 4-20 % (Life Therapeutics) or Tris-Glycine iGels 4-20% (Life Therapeutics), electrophoresis was run at 150V.

Sample Preparation for 2D electrophoresis

20% TCA precipitation: thawed urine was diluted in 90% Trichloro-Acetic acid (TCA) to a final concentration of 20% TCA. Samples were precipitated at -20⁰C for 2 hours, and centrifuged at 2500Og, 4°C for 15 min. Pellet was carefully resuspended using a probe sonicator in 1 ml cold acetone and centrifuged again at 2500Og, 4°C for 15 min. Pellet was air dried and resuspended in the electrophoresis buffer (Miprep F) and frozen at -20⁰C until used for 2DE .

Sample Preparation for ELISA assay

Small interfering compounds were removed from urine samples by using a centrifugal filtration, using Centricon (Millipore) device with a 3 KDa membrane cut-off, as per manufacturer's instructions.

2D electrophoresis

2D electrophoresis was used to separate protein mixtures. The separation, based on isoelectric point for the first dimension and on molecular weight for the second dimension, allowed individual spots on the gel. Each spot corresponded to a specific protein, which was later isolated (spot picked) and digested with trypsin. The peptides of interest are then subjected to mass spectrometry analysis.

IPG Strip Rehydration

Ge/ loading

Depending on the SDS-PAGE results, 2 to 10 ml of sample were precipitated with 20% TCA and the pellet was resuspended in 230 μl of MiPrep F buffer (Minomic Pty Ltd, Australia). This MiPrep F solution was then stored at -20⁰C overnight and used to rehydrate dried 11 cm-IPG strips (pH 4-7, BioRad) overnight.

Iso-Electric Focusing (IEF)

Proteins are amphoteric compounds. The net charge of a protein (either positive, negative or zero) depends on the pH of its local environment. The specific pH at which the net charge of a protein is zero is its pi (isoelectric point). Therefore at its pi, a specific protein stops migrating in an electric field. IEF is thus a technique to separate proteins according to their pi.

Isoelectric focusing was performed overnight at a constant temperature of 2O⁰C by using an IPGphor (Amersham Pharmacia Biotech) under the following conditions: 2 h at 100 V, 4 h at 300 V, 2 h at 600 V, 2 h at 1000 V, 1 h at 2500 V, 1 h at 3,000 V and 5 h at 5,000 V, reaching a total of 35 kVhrs, 100 μA/strip.

Strips were then stored at -20⁰C for at least 2 hours, before running the second dimension.

IPG strip equilibration

Prior to running the second dimension, strips had to be equilibrated with alkylation/reduction buffer (SDS buffer) in order to transform the proteins into SDS- proteins complexes. Then, the proteins will be completely unfolded and will carry negatives charges only. Strips were incubated with reduction buffer for 15 min followed by incubation in alkylation buffer for a further 15 min. SDS-PAGE

Because the proteins are all negatively charged during electrophoresis, SDS- PAGE separates by molecular mass only.

The second-dimension SDS-PAGE separation was performed using Criterion Precast Gel (Tris-HCI, 8-16 % gel, BioRad) in Tris-glycine buffer. For 11-cm IPG strips, separation conditions were 500 mA/gel and 200 V until the bromophenol dye front reached the bottom of the gel (usually 1 hour).

Detecting Proteins Fixing of proteins

Proteins were fixed by agitating the gel for at least 1 hour in 10 % ethanol, 7% acetic acid.

Staining of proteins

Gels were stained with SYPRO Ruby fluorescent stain (Molecular Probe) overnight. SYPRO Ruby staining has a high sensitivity and is Mass Spectrometer compatible.

Destaining

After staining, gels were incubated in 10% ethanol, 7% acetic acid for at least 2 hours in order to wash excess dyes out of the polyacrylamide gel. Ge/ Imaging

Gels were imaged using the ProXpress CCD Imager (PerkinElmer) (480 nm Excitation, 620 nm Emission).

Spot Selection and excision Image analysis

The TIF images generated with ProXpress CCD Imager were aligned using the TT900 alignment software (nonlinear dynamics) to the image chosen as reference gel, and new aligned images were analysed using Progenesis discovery version 2006 (PG240, nonlinear Dynamics), creating two groups containing control and diabetes gels respectively. The reference gel was edited and the samespot function was applied to transfer spot outlines to all remaining gels. Spots present on subgels but missing from the reference gel were manually added to the reference and transferred to subgels. Finally, average gels of each group were recreated to update the spot values, and INCA normalised volumes were compared between the two groups. Spots having a P-value <= 0.05 (student t-Test between diabetes and control populations) were retained and manual examination of histograms, montage window and spot intensities were used as discriminatory parameters in order to select potential biomarkers.

Spot picking

Selected proteins were isolated from SYPRO stained 2D gels, by automated spot excision (ProPic Station, PerkinElmer).

Mass Spectrometry

In Gel tryptic digestion

The protein spots of interest were subjected to tryptic digestion. Trypsin is a highly specific protease, which cuts directly downstream of the two basic amino acids lysine (K) and arginine (R). As long as digestion is complete on one protein, it will produce a set of peptides of varying masses that are characteristic of that protein. The mass of each peptide will be the sum of its amino acids masses including any modification that they have undergone.

For manual digestion, the gel pieces were washed 1 time in 50 % acetonitrile / 50 mM ammonium bicarbonate, and then 1 time in 100 % acetonitrile. The dried gel spots were then rehydrated with 30 ul of digestion buffer (trypsin at

5 ug/ml in 50 mM amonium bicarbonate) and incubated overnight at 37⁰C.

Characterisation of protein spots by Maldi-TOF-MS and Databank searching

After tryptic digest, the samples were sonicated in a water bath for 10 minutes to extract peptides from the gel plugs. Tryptic digests were desalted and concentrated using C18 ZipTips (Millipore) as follows: C18 ZipTip was activated 3 times in 70% ACN, 0.1% TFA, equilibrated 3 times in 0.1% TFA, and 10 ul of sample was loaded. After 3 washes in 0.1% TFA, peptides were eluted on the 96-position target plate using 1.8 μl of matrix (α-cyano-4-hydroxycinnamic acid in 70% ACN, 0.1% TFA, and air-dried before analysis. Peptide mass fingerprint was obtained on a MALDI-TOF MS (Waters), operated in reflectron mode. The peak list was searched against the non-redundant protein database Swiss-Prot using mascot. Swissprot databank was search using Homo sapiens taxonomy parameter, with Trypsin as enzyme and.1 miscleavage allowed. Variable modifications were set to: oxidation of methionine residues and alkylation of cysteine residues by propionamide. A peptide tolerance of 300 ppm was allowed with an upper limit on intact protein mass of 150 KDa.

The excluded masses were 1046.542, 1060.088, 1082.070, 1098.044, 1126.565, 1424.765, 1533.858, 1940.935, 2211.104, 2225.120, 2239.136, 2283.181, 2297.196, 2465.199, 2807.314, 833.090, 842.510, 855.072, 870.541, 871.046, and 907.994.

ELISA immunological assays

Kininogen assay

A 96 well single-Break Strip plate, PS₁ C-bottom high binding, crystal clear (Greiner bio-one) was coated with 100 uL/well of HMW Kininogen Heavy Chain Monoclonal antibody (QED bioscience) at a concentration of 2 ug/ml in Phosphate Buffered Saline (PBS), and left at 4⁰C overnight. Solution was removed and replaced with 200 uL /well of Blocking solution (20% skim milk in PBS), and incubated at 4⁰C overnight (for use up to one week after blocking). Blocking solution was discarded and wells were washed 2 times with 200 uL washing solution (PBS, 0.05% Tween). 10OuL of urine sample dilutions or standard dilutions was added to each well, and incubated 2 hours at 37⁰C. The standard dilutions were made of Kininogen HMW, 2 chains, isolated from human plasma (Sigma) diluted in PBS, 0.1% Bovine Serum Albumin (BSA) to the following concentrations: 0 (Blank); 200; 400; 600; 800; 1000; 1500; 2000 ng/mL The urine sample dilutions were made using pre-processed urine (see Sample preparation for ELISA assay), diluted in PBS, 0.1% BSA according to the following proportions: 1:0 (neat urine); 1:1 and 1:4 (v/v urine sample: PBS, 0.1% BSA). Each sample or standard dilution was loaded in triplicates wells on the ELISA plate.

After incubation, sample and standard solutions were discarded and wells were washed 2 times. 100 ul Sheep anti-human high molecular weight Kininogen antibody, HRP conjugated (Cedarlane) at 2ug/ml in PBS, 0.1% BSA, was added to each well and incubated for 1 hour at 37⁰C. The antibody solution was discarded and the ELISA plate was washed 4 times before adding 100 uL/well of 3,3',5,5' Tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma). The plate was incubated in the dark for 10 minutes before adding 100ul/well 1 M sulfuric acid to stop the enzymatic reaction, and absorbance was read at 450 nm.

A 4-parameter curve was fitted to the standard curve using SOFTmax PRO (version 3.0) software, from which Kininogen concentrations of the sample dilutions were automatically calculated. Only the sample dilutions having an absorbance in the most sensitive range of the standard curve (linear section of the 4-parameter standard curve) were taken into account to average the Kininogen concentration in the original urine sample.

Serotransferrin assay

A 96 well single-Break Strip plate, PS, C-bottom high binding, crystal clear (Greiner bio-one) was coated with 100 uL/well of Mouse monoclonal [1C10] antibody to Transferrin Abeam) at a concentration of 2 ug/ml in Phosphate Buffered Saline (PBS), and left at 4⁰C overnight. Solution was removed and replaced with 200 uL /well of Blocking solution (20% skim milk in PBS), and incubated at 4⁰C overnight (for use up to one week after blocking). Blocking solution was discarded and wells were washed 2 times with 200 uL washing solution (PBS, 0.05%Tween). 10OuL of urine sample dilutions or standard dilutions were added to each well, and incubated for 2hours at 37⁰C. The standard dilutions were made of human transferrin (Invitrogen) diluted in PBS, 0.1% BSA to the following concentrations: 0 (Blank); 15; 30; 50; 70; 100; 300; 500 ng/mL. The urine sample dilutions were made using pre-processed urine (see Sample preparation for ELISA assay), diluted in PBS, 0.1% BSA according to the following proportions: 1:1 ; 1 :4 and 1 :9 (v/v urine sample: PBS, 0.1% BSA). Each sample or standard dilution was loaded in triplicates wells on the ELISA plate. After incubation, sample and standard solutions were discarded and wells were washed 2 times. 100 ul Rabbit polyclonal antibody to Transferrin, HRP conjugated (Abeam) (Cedarlane) at 50 ng/ml in PBS, 0.1% BSA, was added to each well and incubated for 1 hour at 37⁰C. The antibody solution was discarded and the ELISA plate was washed 4 times before adding 100 uL/well of 3,3',5,5' Tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma). The plate was incubated in the dark for 30 minutes before adding 100ul/well 1 M sulfuric acid to stop the enzymatic reaction, and absorbance was read at 450 nm.

A 4-parameter curve was fitted to the standard curve using SOFTmax PRO (version 3.0) software, from which the Transferrin concentration of the sample dilutions were automatically calculated.

Only the sample dilutions having an absorbance in the most sensitive range of the standard curve (linear section of the 4-parameter standard curve) were taken into account to average the Serotransferrin concentration in the original urine sample. RESULTS

Identification of differentially expressed proteins in control and diabetic samples

Urine is a challenging sample due to high inter- and intra-individual variability, low protein concentration and high salt content, often requiring extensive sample preparation.

A fast and efficient method was employed where urine samples were precipitated with 20% Trichloroacetic Acid (TCA) to desalt and increase the amount of proteins loaded on 2D electrophoresis gels.

Urine samples from 7 type Il diabetic and 10 healthy control people were collected, concentrated and desalted prior to the display of proteins spots on 2D gel electrophoresis and staining with SYPRO Ruby. Figure 1 and Figure 2 show 2D gel electrophoresis of control and diabetic urine samples respectively.

2DE images were aligned to a chosen reference gel using TT900 software (NonLinear Dynamics), allowing accurate spot matching across all the gels. Aligned images were then compared using Progenesis PG240 Image Analysis software (NonLinear Dynamics). After grouping images as either Diabetes or Control, spot editing was performed on the reference gel only. The Samespot function was applied, transferring spot outlines from the reference to all remaining subgels. Spots on subgels that were absent from the reference gel were manually added and transferred to all remaining gels. As a result, 891 spots appeared on each gel (see Figure 3). This image analysis method allows very efficient automatic spot matching and more reliable statistical analysis by avoiding missing values.

After background subtraction and INCA normalisation, average gels were created for Diabetes and Control groups in order to compare average intensities of spots between the 2 groups.

Potential biomarkers were selected according to the following criteria:

• Student T-test: p-value (INCA normalised volume, Diabetes group, Control group) <= 0.05

• INCA normalised volume histogram showing different expression pattern between groups

, • Montage window showing different intensities between groups • • Preference for groups of spots rather than isolated spots

• Single spots with weak intensity were rejected Using this selection, 16 spots were found over-expressed and 21 under- expressed in type Il diabetes. Interestingly, many of them were grouped in "trains" typical of post-translational modifications (PTM) (see Figure 4).

In order to identify selected biomarkers, spots were excised using the proExcision robot (PerkinElmer), digested with trypsin and fractionated by affinity chromatography using C18 Ziptip (Millipore), allowing desalting and concentration of peptides prior to spotting on a Maldi-TOF target plate.

MALDI-TOF (Waters) was run in reflectron mode followed by manual Swissprot databank searching using Mascot search engine to identify the digested proteins. Published urinary proteome maps were also used in order to confirm or establish protein identification. This allowed identification of selected biomarkers, two of them over- expressed and four of them under-expressed in type Il diabetes as shown on Figure 4.

Table 1 shows the protein identities of the spots shown on Figure 4.

Development and optimisation of ELISA immu no-assay to measure biomarkers amount in urine.

Commercially available antibodies specific to human Kininogen or human Transferrin were purchased in order to optimise a sandwich ELISA assay for each of those two biomarkers, suitable to urine samples.

Urine samples needed pre-processing in order to remove small compounds interfering with ELISA. Blocking solution and incubation time were optimised in order to minimise the background level. For each biomarker, concentrations of capture and detection antibodies, as well as standard dilutions and enzymatic reaction time were optimised in order to obtain a standard curve covering the totality of the sensitivity range of the assay (typically S-shapped curve), within the detection limits of the plate reader instrument. \

The optimised concentrations and enzymatic reaction time are presented in the methods. Figure 5 presents such optimised standard curves for both Kininogen and Transferrin ELISA assays.

The assays were applied to pre-processed urine samples, and only sample dilutions having an absorbance value within the sensitivity range of the standard curve were retained. Both Intra-assay and Inter-assay variation (measured as CV) were bellow 10% for each assay, and biomarker concentrations were consistent between dilutions of a same urine sample.

The present inventors have developed two sensitive and reproducible assays, allowing measurement of Transferrin and High Molecular Weight Kininogen in human urine samples to detect diabetes.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. REFERENCES

Heine G., Raida M. and ForssmannW-G. (1997) Mapping of peptides and proteins fragments in human urine using liquid chromatography-mass spectrometry, Journal of Chromatography, 776, 117-124. Spahr C.S., Davis M. T., McGinley M.D., Robinson J.H., Bures E.J., Beierle

J., Mort J., Courchesne P.L., Chen K., Wahl R.C., Yu W., Luethy R., Scott D. and Patterson S.D. (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I. Profiling an unfractionated tryptic digest, Proteomics, 1, 93-107. Thongboonkerd V., McLeish K.R., Arthur J.M. and Klein J.B. (2002),

Proteomic analysis of normal urinary proteins isolated by acetone precipitation or ultracentrifugation, Kidney International, 62, 1461-1469.

Koivunen, M. E., Dettmer, K., Vermeulen, R., Bakke, B., et al.(2006),

Improved methods for urinary atrazine mercapturate analysis — Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE), Analytica Chimica Acta, 572, 180-189.

Claims

Claims:

1. An assay for testing diabetes or a predisposition to diabetes in a subject comprising: analysing a biological fluid from a subject for the presence of one or more biomarkers selected from the group consisting of SEQ ID NO:1 , SEQ ID NO: 2,

SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or respective isoforms, peptides and fragments thereof, wherein detection of the biomarker is indicative of diabetes or a predisposition to diabetes in the subject.

2. The assay according to claim 1 , wherein the diabetes is Type Il diabetes.

3. The assay according to claim 1 or 2, wherein the biological fluid is selected from the group consisting of urine, saliva, blood, blood products such as serum, plasma, tears, cerebrospinal fluid, and lymph.

4. The assay according to claim 3, wherein the biological fluid is urine.

5. The assay according to any one of claims 1 to 4, wherein proteins present in the biological sample are digested to form peptide fragments which are detected by conducting mass spectrophotometric analysis on the digested sample to elucidate discernible peptide fragments contained therein; and comparing mass spectrum profiles of peptides forming Serotransferrin [Precursor], Kininogen [Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor],

Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2- glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 -microglobulin [Precursor] or respective isoforms to mass spectrum profiles of peptides elucidated from the sample; wherein recognition of a mass spectrum profile in the sample displaying the mass spectrum profile for any one or more of proteins or peptides having amino acid residues selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or isoforms thereof is indicative of diabetes or a predisposition to diabetes.

6. The assay according to any one of claims 1 to 5, wherein the subject is a human.

7. The assay according to any one of claims 3 to 6, wherein the urine is concentrated by trichloroacetic acid precipitation.

8. The assay according to any one of claims 3 to 6, wherein the urine is concentrated by precipitation using acetone or membrane-based separation.

9. The assay according to any one of claims 1 to 8, further comprising separating the proteins, peptides or fragments in the biological fluid by chromatography.

10. The assay according to claim 9, wherein the separated proteins, peptides or fragments are identified by mass spectrometry.

11. The assay according to claim 10, wherein the proteins, peptides or fragments are about 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more amino acids in length.

12. The assay according to any one of claims 1 to 11 , further comprising: obtaining a biological sample from the subject; measuring the concentration of creatinine in the sample; digesting the proteins present in the sample to form peptides; and optionally, separating the peptides.

13. An isolated antibody directed to a protein, protein fragment or peptide detectable in a biological sample of a subject being indicative of diabetes or a predisposition to diabetes in a subject, wherein the protein, protein fragment or peptide is selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or isoforms thereof.

14. The antibody according to claim 13, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

15. The antibody according to any one of claims 13 or 14, wherein the antibody is detectably labelled.

16. The antibody according to claim 15, wherein the label is fluoresein isothiocyanate (FITC), Texas Red, Oregon Green, TRITC, Alexa dyes, allophycocyanin or rhodamine.

17. The antibody according to claim 16, wherein the label is a radiolabel.

18. A kit for assaying a subject for diabetes or a predisposition to diabetes comprising: one or more antibodies according to any one of claims 13 to 17; and suitable reagents and diluents for carrying out a diabetes assay.

19. Use of a biomarker selected from Serotransferrin [Precursor], Kininogen

[Precursor], Polymeric-lmmunoglobulin Receptor [Precursor], Alpha-1 -antitrypsin [Precursor], Monocyte differenciation antigen CD14 [Precursor], Leucine-rich alpha-2-glycoprotein [Precursor], Deoxyribonuclease 1 [Precursor], Alpha-1 - microglobulin [Precursor] or respective isoforms or peptides or fragments thereof in an assay for diabetes or a predisposition to diabetes.