WO2011071368A1 - Method for measuring hepcidin - Google Patents

Method for measuring hepcidin Download PDF

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Publication number
WO2011071368A1
WO2011071368A1 PCT/NL2009/050759 NL2009050759W WO2011071368A1 WO 2011071368 A1 WO2011071368 A1 WO 2011071368A1 NL 2009050759 W NL2009050759 W NL 2009050759W WO 2011071368 A1 WO2011071368 A1 WO 2011071368A1
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Prior art keywords
hepcidin
chicken
antibodies
antibody
rabbit
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PCT/NL2009/050759
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French (fr)
Inventor
Nicolai Ilych Grebenchtchikov
Cornelus Godefridus Jacobus Sweep
Dorine Wilhelmine Swinkels
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Umc St. Radboud
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Priority to PCT/NL2009/050759 priority Critical patent/WO2011071368A1/en
Publication of WO2011071368A1 publication Critical patent/WO2011071368A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates to assays for determining hepcidin in biological samples and in particular to enzyme-linked immunosorbent assays (ELISA) therefore.
  • ELISA enzyme-linked immunosorbent assays
  • Hepcidin is a circulating peptide hormone that regulates the iron absorption, uptake and mobilization from stores to meet body iron need. It is primarily secreted by hepatocytes and is highly conserved among different species.
  • the mature bioactive form of hepcidin is a 25 amino- acid peptide that derives from a precursor of 84 amino acids that undergoes 2 enzymatic cleavages.
  • Other isoforms that exist in human serum and urine are the N-terminal truncated hepcidin-20 and -22 peptides that consist of 20 and 22 amino acids, respectively. These isoforms are without apparent biological function, although in vitro at supraphysiologic concentrations, hepcidin-20 shows broad spectrum antimicrobial activity. By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake and mobilization from stores to meet body iron need.
  • hepcidin Since the discovery of hepcidin and the demonstration of its crucial role in iron homeostasis, there has been a substantial interest in developing a reliable assay of the peptide hormone in body fluids. Measurement of hepcidin levels in serum is thought to improve understanding of disorders of iron metabolism. Furthermore, hepcidin dosage is considered to be a useful tool in the differential diagnosis and clinical management of these diseases. Certain physiologic and pathologic processes regulate the synthesis of hepcidin. Situations in which demand for circulating iron is increased (particularly erythropoietic activity) bring about a decrease in hepcidin synthesis. These include iron deficiency, hypoxia, anemia, conditions characterized by ineffective erythropoiesis (e.g.
  • hemoglobinopathies or the use of erythropoiesis stimulating agents.
  • the decrease in hepcidin results in the release of stored iron and an increase in dietary iron absorption.
  • infection or inflammation cause an increase in hepcidin synthesis, which causes a decrease in circulating iron, thought to protect the body from extracellular proliferating pathogens.
  • WO 2009/058797 concerns the production of monoclonal antibodies throug expression of aan appropriate nucleotide sequence in a host such as Chinese hamster ovary cells. The use of these monoclonal antibodies in immunoassays is mentioned.
  • WO 2008/097461 concerns the preparation and isolation of monoclonal antibodies against correctly folded human hepcidin.
  • the monoclonal antibodies were obtained from immunisation of mice and rats.
  • a sandwich ELISA is described using IgG from polyclonal rabbit antisera. The sensitivity of the assay was found to be low which was explained by that the ability of two antibodies to bind simultaneously to hepcidin may be a rare event. Summary of the invention
  • An objective of the present invention is to provide an assay for measuring hepcidin, in particular to be able to accurately determine the concentration of hepcidin in biological samples such as blood and urine, with maximum sensitivity.
  • the assay can be easily and routinely applied in laboratories, e.g. within a clinical setting without the need of sophisticated equipment such as mass spectrometers or taking other special measures, e.g. working with isotopes.
  • immunological assays for measuring and quantifying hepcidin in human subjects there is clearly the tendency to move towards using monoclonal antibodies for optimal binding to hepcidin.
  • a standard competitive ELISA gave an unprecedented analytical sensitivity when polyclonal antibodies generated in chicken were used.
  • Hepcidin-25 the mature, bioactive isoform as present in humans was used for immunization of the chicken.
  • rabbits are the animals of choice for generating polyclonal antibodies against hepcidin
  • chicken polyclonal antibodies detection levels that are 2 orders of magnitude lower could be measured in a competitive ELISA.
  • a further improvement in sensitivity could be obtained by performing a sandwich ELISA which involves, after the binding of hepcidin by chicken polyclonal antibodies, contacting the resulting hepcidin-chicken antibody complex with polyclonal antibodies generated in rabbits.
  • the analytical sensitivity improved a further order of magnitude.
  • the mature, bioactive isoform hepcidin-25 present in humans was used for immunization of the chicken.
  • the first sandwich ELISA for measuring hepcidin is provided, based on polyclonal antibodies from the two different animals chicken and rabbits. It is considered that the present 2-site immunoassay specifically recognizes the hepcidin- 25 isoform. This overcomes the major drawback of currently available one-site immunoassays which cannot discriminate between the naturally available isoforms.
  • the present invention thus concerns a method for determining the amount of hepcidin in a sample comprising a) obtaining a sample to be assayed for human hepcidin, b) incubating the sample with polyclonal antibodies from chicken immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay.
  • the sample to be assayed is preferably obtained from a human or preferably is isolated from a human.
  • a sample can be any biological sample from a human wherein hepcidin that is to be measured can be expected.
  • the biological sample is a sample wherein the concentration of hepcidin can be correlated to a physiological condition or disorder or is relevant for diagnosis.
  • the sample is obtained from a biological fluid, e.g. bodily liquor, and in a preferred embodiment the sample is isolated or derived from plasma, serum or urine of a human. In another embodiment the sample may be obtained from a culture supernatant of hepcidin producing cells.
  • the hepcidin that is used for immunisation of chicken preferably is human hepcidin-25.
  • the human hepcidin-25 that is used for immunization can be prepared by any method such as via recombinant expression technology or via synthesis.
  • chickens at start immunization are immunized intramuscularly (pectoral) with synthetic hepcidin, emulsified in an equal volume of Freund's complete adjuvant.
  • Booster inoculations with a similar amount of hepcidin emulsified in Freund's incomplete adjuvant are administered every two weeks.
  • About 6 weeks after the start immunization eggs from immunized chickens are collected daily.
  • a suitable amount of hepcidin to use at start immunization is about 10-15 ⁇ g in 250 ⁇ PBS.
  • IgY fraction of egg yolk is isolated. Isolation and purification of IgY is known in the art, e.g. Polsen et al. Immunol Invest (1985), 14:323-327. Firstly, IgY is isolated form egg yolk by sequential precipitation in increasing PEG concentration. Then IgY is purified by affinity chromatography. Suitably IgY is purified by affinity chromatography on hepcidin-24 coupled columns.
  • a competitive ELISA is performed.
  • the amount of hepcidin bound to chicken polyclonal antibody is measured.
  • the chicken anti- hepcidin antibodies may either be labelled or unlabeled. Unlabeled antibodies are preferably used in combination with labelled detection reagents such as those that bind to the immunocomplexes, e.g.
  • anti-chicken antibodies or other secondary antibodies or antigen-binding fragments thereof capable of binding to the chicken polyclonal antibodies that bind to the hepcidin, or in a competitive assay, labelled detection reagents, in particular labelled hepcidin, that are bound by the chicken polyclonal antibodies.
  • the labelled hepcidin may be detectable through binding of a specific binding partner, e.g. a biotin/avidin - streptavidin binding pair comprising a further detectable label, for example an indirect label.
  • Indirect labels that can be indirectly detected by their production of a detectable reaction product include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product.
  • An improvement of the present method for determining the amount of hepcidin was achieved by including a subsequent step of incubating binding antibodies to allow binding to the hepcidin bound to the chicken antibodies. In such a sandwich ELISA the antibodies used for incubation in the subsequent step are rabbit polyclonal antibodies.
  • the method comprises after step b) the step of contacting the incubated chicken antibodies with polyclonal antibodies from rabbit immunized with hepcidin that allow binding of polyclonal rabbit antibodies to the hepcidin bound to the chicken antibodies.
  • the present invention thus also concerns a method for determining the amount of hepcidin in a sample comprising a) obtaining a sample to be assayed for human hepcidin, bl) incubating the sample with polyclonal antibodies from chicken immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample, b2) contacting the incubated chicken antibodies with polyclonal antibodies from rabbit immunized with hepcidin that allow binding of polyclonal rabbit antibodies to the hepcidin bound to the chicken antibodies and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay.
  • the hepcidin that is used for immunisation of rabbits is preferably the same as that is used for immunization of chicken which preferably is human hepcidin-25.
  • the human hepcidin-25 that is used for immunization can be prepared by any method such as via recombinant expression technology or via synthesis.
  • rabbits at start immunization are immunized with synthetic hepcidin, emulsified in an equal volume of Freund's complete adjuvant. Injections can be subcutaneously, but advantageously the first injection is in the popliteal gland.
  • Booster inoculations with a similar amount of hepcidin emulsified in Freund's incomplete adjuvant are administered subcutaneously.
  • Injections may be repeated every two weeks but advantageously injections are repeated at 4-week intervals. Injections are repeated for example 5-15 times, e.g. about 10 times. Just before each injection blood, about 5 or 6 ml, is collected. A suitable amount of hepcidin to use at start immunization is about 10-30 ⁇ g in 250-500 ⁇ PBS.
  • polyclonal antibodies against hepcidin were purified by affinity chromatography.
  • rabbit anti-hepcidin IgG is purified by affinity chromatography on hepcidin-24 coupled columns.
  • binding of the rabbit antibodies is preferably carried out in a non-competitive assay.
  • step c) the amount of rabbit antibody that is bound to hepcidin that is bound by chicken antibody is measured.
  • the rabbit anti-hepcidin antibodies may either be labelled or unlabeled.
  • the rabbit anti-hepcidin antibodies may be determined by a label that can be indirectly detected by the production of a detectable reaction product including various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product.
  • enzymes well known in the art such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product.
  • a preferred method for carrying out the immunoassay according to the invention involves immobilising the chicken polyclonal antibody on a solid support.
  • this immobilisation is carried out directly onto the solid support itself, e.g. a microtiter plate, such immobilisation may lead to conformational changes of the antibody and may also lead to less reproducibility. Therefore in a preferred embodiment, the present method comprises binding the chicken polyclonal antibody to anti-chicken antibody which is immobilized on a solid support.
  • the anti-chicken antibody is duck anti-chicken antibody which minimises disturbances.
  • Duck anti-chicken antibody can be prepared as described in Grebenchtchikov et al. (2002), 268:219-231.
  • the rabbit antibodies are detected by anti-rabbit antibody.
  • the anti-rabbit antibody is labelled.
  • the anti-rabbit antibody is goat anti-rabbit antibody.
  • the anti-rabbit antibody may be determined by a label that can be indirectly detected by the production of a detectable reaction product including various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product.
  • the label is horseradish peroxidase.
  • the order of incubating with polyclonal antibodies in the sandwich-type ELISA may be altered, thus steps bl) and b2) may be switched.
  • the present method comprises a) obtaining a sample to be assayed for human hepcidin, bl) incubating the sample with polyclonal antibodies from rabbit immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample, b2) contacting the incubated rabbit antibodies with polyclonal antibodies from chicken immunized with hepcidin that allow binding of polyclonal chicken antibodies to the hepcidin bound to the rabbit antibodies and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay.
  • the present method is for determining the amount of human hepcidin-25. In another embodiment the present method is for determining the amount of hepcidin highly homologous to human hepcidin-25, in particular for Cynomologus Monkey hepcidin.
  • the invention in another embodiment relates to a kit of parts for carrying out the method according to the present invention comprising anti hepcidin polyclonal chicken antibody, and preferably also anti hepcidin polyclonal rabbit antibody, and preferably also duck anti-chicken antibody and labeled goat anti-rabbit antibody or labeled duck anti-chicken antibody and goat anti-rabbit antibody.
  • the kit of parts comprises anti hepcidin polyclonal chicken antibody, anti hepcidin polyclonal rabbit antibody, duck anti-chicken antibody and labelled goat anti-rabbit antibody.
  • Chickens were initially immunized intramuscularly (pectoral) with synthetic hepcidin -25 (10-15 ⁇ g in 250 ⁇ PBS), emulsified with an equal volume of Freund's complete adjuvant.
  • Booster inoculations containing 10-15 ⁇ g immunogen prepared with Freund's incomplete adjuvant, were administered every two weeks. Approximately 42 days after start immunization eggs from the immunized chickens were collected daily (collection).
  • Isolation of IgY fraction from the egg yolk and affinity purification of the polyclonal antibodies were performed as previously described(Polsen et al. Immunol Invest (1985), 14:323-327.): After separation of egg-yolk from egg-white and intensive flushing with water, the yolk membrane was pierced and the yolk content was collected in a tube. The volume was adjusted to 30 ml with PBS and the solution obtained was chilled on ice. Subsequently 30 ml of 7% PEG in PBS (final concentration 3.5%) was added and the mixture was left for 10 min at 4°C. The mixture was then centrifuged at 5,000xg for 20 min at 4°C. To the supernatant (ca.
  • Polyclonal anitbodies against hepcidin were purified by affinity chromatography on hepcidin-24 coupled columns (Affi-Gel® 10/15; Bio-Rad Laboratories, Hercules, CA, USA). Before being applied to the columns the samples of antibodies (ca. 4 ml) were filtrated and thereafter passed through the columns three times. The elution of antibodies from the columns was performed after the washing with 50 ml of PBS. The chicken antibodies were eluted with 8 ml of 5.0 mol/L KSCN in 50% ethylene glycol/water. Antibodies were stored in aliquots at -20°C
  • Synthetic human hepcidin was used for immunizing rabbits. Aliquots containing 10 ⁇ g hepcidin- 25 in 500 ⁇ PBS were mixed with an equal volume of Freund's complete adjuvant for the first injection and with Freund's incomplete adjuvant for boosters. The first injection was in the popliteal gland, whereas booster inoculations were administered subcutaneously. Injections were repeated nine times at 4-weekintervals. Blood (5 ml) was collected just before the injections in citrate tubes. After centrifugation the citrate plasma was stored at -20°C.
  • Polyclonal antibodies against hepcidin were purified by affinity chromatography on hepcidin-24 coupled columns (Affi-Gel® 10/15; Bio-Rad Laboratories, Hercules, CA, USA). Before being applied to the columns the samples of antibodies (ca. 4 ml) were filtrated and thereafter passed through the columns three times. The elution of antibodies from the columns was performed after the washing with 50 ml of PBS. The rabbit antibodies were eluted with 8 ml of PBS containing 1.25 mol/L KSCN.
  • the purified rabbit anti-hepcidin antibodies were concentrated approximately 10 times by Amicon Ultra- 15 centrifugal filter device (Millipore, Amsterdam, the Netherlands) and, after dilution with glycerol (1 :1), stored at -20°C.
  • Hepcidin-25 was biotinylated by incubating 1 1 .5 ⁇ g hepcidin-25 (Peptide International, Louisville, KY, USA) with 8.17 ⁇ g No-WeighTM Sulfo-NHS-Biotin (20-fold molar excess) (Pierce, Rockford, IL, USA) during 30 minutes at ambient temperature.
  • the procedure started with coating of the microtiter plate (96-well Nunc MaxisorbTM flat bottomed) with 100 ⁇ of duck-anti-chicken IgY antibody (Grebenchtchikov et al. (2002), 268:219-231) (4 mg/L in 50 mmol/L NaHC03/Na 2 C03, pH 9.6) overnight at 4°C.
  • the plate was then washed four times with 300 ⁇ of washing buffer (1 mL/L Tween-20 in PBS) by use of a 96PW plate washer (Tecan, Mannedorf, Switzerland).
  • the plates were blocked with 10 g L BSA (Sigma Aldrich, St. Louis, MO, USA) in PBS (300 ⁇ ⁇ ) for 2h at ambient temperature and washed four times with washing buffer.
  • the next step was the incubation with chicken-anti-hepcidin antibody [100 ⁇ ⁇ ⁇ ; 0.3 mg/L in dilution buffer (10 g/L BSA in washing buffer)] for 2h at ambient temperature.
  • the calibrator hepcidin-25
  • the unknown samples (20-fold diluted in dilution buffer unless stated otherwise
  • the reference sample 75 ⁇ ⁇ in dilution buffer
  • 75 ⁇ ⁇ biotinylated hepcidin dilution 10,000x
  • the plates were incubated overnight at 4°C.
  • the plates were washed four times with 300 ⁇ washing buffer and subsequently plates were incubated with 100 ⁇ /well Streptavidin-POD conjugate (peroxidase conjugated to streptavidin; Roche Diagnostics GmbH, Penzberg, Germany) (dilution 50,000x) during lh at ambient temperature.
  • the sandwich structure employed includes four different antibodies (Abs), a coating Ab (duck anti-chicken IgY) a capture Ab (chicken anti-hepcidin), a trapping Ab (rabbit anti-hepcidin) and finally a detection Ab (HRP-labeled goat anti-rabbit).
  • the assay is insensitive to heterophilic antibodies. All incubation steps were separated by washing steps (4 times, 300 ⁇ of washing buffer per well).
  • the coating Ab was diluted in the coating buffer, whereas capture, trapping and detecting Abs as well as standards, unknowns and reference sample were diluted in the dilution buffer (100 ⁇ per well).
  • microtiter plates were first overnight coated at 4°C with coating antibody (duck anti-chicken IgY) antibody, and then treated for 2 hours at 37°C with blocking buffer. Next, the plate was incubated for 2 hours at RT with capture antibody (chicken anti-hepcidin) and the samples, reference samples and standards were incubated overnight at 4°C.
  • Trapping antibody (rabbit anti- hepcidin) and detection antibody (HRP-labeled goat anti-rabbit, commercially available from Thermo Fisher Scientific, Pierce Protein Research Products, Rockford, IL, USA) were sequentially incubated for 2 hours at RT and plates were developed with substrate solution in the dark for 30 min at RT, color reaction was stopped with H 2 SO 4 , and optical density was measured at 492 nm within 30 minutes.

Abstract

An improved immunological assay for quantifying human hepcidin is provided involving the use of chicken polyclonal antibodies. In particular a 2-site sandwich-type ELISA wherein chicken polyclonal antibodies and rabbit polyclonal antibodies bind to hepcidin show unprecedented sensitivity.

Description

Method for measuring hepcidin
Field of the invention
The present invention relates to assays for determining hepcidin in biological samples and in particular to enzyme-linked immunosorbent assays (ELISA) therefore.
Background of the invention
Hepcidin is a circulating peptide hormone that regulates the iron absorption, uptake and mobilization from stores to meet body iron need. It is primarily secreted by hepatocytes and is highly conserved among different species. The mature bioactive form of hepcidin is a 25 amino- acid peptide that derives from a precursor of 84 amino acids that undergoes 2 enzymatic cleavages. Other isoforms that exist in human serum and urine are the N-terminal truncated hepcidin-20 and -22 peptides that consist of 20 and 22 amino acids, respectively. These isoforms are without apparent biological function, although in vitro at supraphysiologic concentrations, hepcidin-20 shows broad spectrum antimicrobial activity. By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake and mobilization from stores to meet body iron need.
Since the discovery of hepcidin and the demonstration of its crucial role in iron homeostasis, there has been a substantial interest in developing a reliable assay of the peptide hormone in body fluids. Measurement of hepcidin levels in serum is thought to improve understanding of disorders of iron metabolism. Furthermore, hepcidin dosage is considered to be a useful tool in the differential diagnosis and clinical management of these diseases. Certain physiologic and pathologic processes regulate the synthesis of hepcidin. Situations in which demand for circulating iron is increased (particularly erythropoietic activity) bring about a decrease in hepcidin synthesis. These include iron deficiency, hypoxia, anemia, conditions characterized by ineffective erythropoiesis (e.g. hemoglobinopathies), or the use of erythropoiesis stimulating agents. The decrease in hepcidin results in the release of stored iron and an increase in dietary iron absorption. On the other hand, infection or inflammation cause an increase in hepcidin synthesis, which causes a decrease in circulating iron, thought to protect the body from extracellular proliferating pathogens.
Thus reliable assays to measure hepcidin in human is called for. Until recently only few investigative tools were available to measure hepcidin in biological fluids, the most reliable thereof being based on mass spectrometry (MS). Where MS based assays require relatively expensive equipment, they have the advantage of distinguishing between the three hepcidin isoforms, hepcidin-25, -22 and -20. Immunoassays on the other hand have the potential of more widespread use among clinical laboratories. However, these assays will measure total hepcidin levels, with (depending on the specificity of the antibody) different contributions from each of the isoforms. Thus progress in developing conventional immunochemical hepcidin assays has been hampered by difficulty in generating specific anti-hepcidin antibodies.
Recently, Ganz et al. (2008) Blood, 1 12:4292-4297, reported an immunoassay for human serum hepcidin by means of a competitive enzyme-linked immmunosorbent assay (C-ELISA). The lower detection limit was reported to be 5.5 μg/L.
Koliaraki et al. (2009) PLoS ONE 4:e4581 reported a competion ELISA using recombinant hepcidin25-His and rabbit polyclonal antibodies raised against this recombinant hepcidin25-His. The detection limit was found to be 5.4 μg/L.
Mamalaki and Koliaraki in Haematologica (2009) 94 (supplment 2), page 507 abstract 1275 descibe steps towards the development of a specific sandwich ELISA for measuring hepcidin in serum using polyclonal antiserum from rabbits after immunisation with recombinant hepcidin25- His and monoclonal antibodies obtained from mice after immunisation with recombinant hepcidin25-His.
Grebenchtchikov et al. Br J Haematol. (2009), 146 : 3 17-325, described a competitive radioimmunoassay (RIA) using polyclonal antibodies elicited against hepcidin in rabbits. US 2006/0019339 studies bass (Morone chrysops) hepcidin and provides a process for oxidative refolding in a bioactive form. For measurement of the bass hepcidin, a competitive ELISA using rabbit polyclonal antibodies is described. US 2007/0224186 discloses monoclonal antibodies directed against a specific fragment of hepcidin25. In order to achieve this mice were immunised with an 8 -amino acid fragment of hepcidin. A competitive ELISa using an isolated monoclonal antibody was described. The possibility of generating poyclonal antibodies is mentioned. WO 2009/058797 concerns the production of monoclonal antibodies throug expression of aan appropriate nucleotide sequence in a host such as Chinese hamster ovary cells. The use of these monoclonal antibodies in immunoassays is mentioned.
WO 2008/097461 concerns the preparation and isolation of monoclonal antibodies against correctly folded human hepcidin. The monoclonal antibodies were obtained from immunisation of mice and rats. In example 29 a sandwich ELISA is described using IgG from polyclonal rabbit antisera. The sensitivity of the assay was found to be low which was explained by that the ability of two antibodies to bind simultaneously to hepcidin may be a rare event. Summary of the invention
An objective of the present invention is to provide an assay for measuring hepcidin, in particular to be able to accurately determine the concentration of hepcidin in biological samples such as blood and urine, with maximum sensitivity. Preferably the assay can be easily and routinely applied in laboratories, e.g. within a clinical setting without the need of sophisticated equipment such as mass spectrometers or taking other special measures, e.g. working with isotopes. When it comes to immunological assays for measuring and quantifying hepcidin in human subjects, there is clearly the tendency to move towards using monoclonal antibodies for optimal binding to hepcidin. However, surprisingly it has now been found that a standard competitive ELISA gave an unprecedented analytical sensitivity when polyclonal antibodies generated in chicken were used. Hepcidin-25 , the mature, bioactive isoform as present in humans was used for immunization of the chicken. Whereas apparently rabbits are the animals of choice for generating polyclonal antibodies against hepcidin, it was unexpected that with chicken polyclonal antibodies detection levels that are 2 orders of magnitude lower could be measured in a competitive ELISA. It was even more surprising that a further improvement in sensitivity could be obtained by performing a sandwich ELISA which involves, after the binding of hepcidin by chicken polyclonal antibodies, contacting the resulting hepcidin-chicken antibody complex with polyclonal antibodies generated in rabbits. The analytical sensitivity improved a further order of magnitude. Also for immunizing rabbits, the mature, bioactive isoform hepcidin-25 present in humans, was used.
Thus despite the small size of hepcidin and the consequent limited accessibility of different epitopes, an extremely sensitive sandwich ELISA has been developed without having to resort to preparing and isolating monoclonal antibodies selected for their optimum binding characteristics to hepcidin. Thus according to the present invention, the first sandwich ELISA for measuring hepcidin is provided, based on polyclonal antibodies from the two different animals chicken and rabbits. It is considered that the present 2-site immunoassay specifically recognizes the hepcidin- 25 isoform. This overcomes the major drawback of currently available one-site immunoassays which cannot discriminate between the naturally available isoforms.
Detailed description
The present invention thus concerns a method for determining the amount of hepcidin in a sample comprising a) obtaining a sample to be assayed for human hepcidin, b) incubating the sample with polyclonal antibodies from chicken immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay.
The sample to be assayed is preferably obtained from a human or preferably is isolated from a human. Such a sample can be any biological sample from a human wherein hepcidin that is to be measured can be expected. Preferably the biological sample is a sample wherein the concentration of hepcidin can be correlated to a physiological condition or disorder or is relevant for diagnosis. Preferably the sample is obtained from a biological fluid, e.g. bodily liquor, and in a preferred embodiment the sample is isolated or derived from plasma, serum or urine of a human. In another embodiment the sample may be obtained from a culture supernatant of hepcidin producing cells.
The hepcidin that is used for immunisation of chicken preferably is human hepcidin-25. The human hepcidin-25 that is used for immunization can be prepared by any method such as via recombinant expression technology or via synthesis.
In a suitable immunization protocol chickens at start immunization are immunized intramuscularly (pectoral) with synthetic hepcidin, emulsified in an equal volume of Freund's complete adjuvant. Booster inoculations with a similar amount of hepcidin emulsified in Freund's incomplete adjuvant are administered every two weeks. About 6 weeks after the start immunization eggs from immunized chickens are collected daily. A suitable amount of hepcidin to use at start immunization is about 10-15 μg in 250 μΐ PBS.
Polyclonal antibodies are then isolated from the egg yolk. Preferably the IgY fraction of egg yolk is isolated. Isolation and purification of IgY is known in the art, e.g. Polsen et al. Immunol Invest (1985), 14:323-327. Firstly, IgY is isolated form egg yolk by sequential precipitation in increasing PEG concentration. Then IgY is purified by affinity chromatography. Suitably IgY is purified by affinity chromatography on hepcidin-24 coupled columns.
In order to be able to accurately quantify hepcidin concentrations in samples, in a preferred embodiment a competitive ELISA is performed. Preferably in step c) the amount of hepcidin bound to chicken polyclonal antibody is measured. For detection purposes, the chicken anti- hepcidin antibodies may either be labelled or unlabeled. Unlabeled antibodies are preferably used in combination with labelled detection reagents such as those that bind to the immunocomplexes, e.g. anti-chicken antibodies or other secondary antibodies or antigen-binding fragments thereof, capable of binding to the chicken polyclonal antibodies that bind to the hepcidin, or in a competitive assay, labelled detection reagents, in particular labelled hepcidin, that are bound by the chicken polyclonal antibodies. For example the labelled hepcidin may be detectable through binding of a specific binding partner, e.g. a biotin/avidin - streptavidin binding pair comprising a further detectable label, for example an indirect label. Indirect labels that can be indirectly detected by their production of a detectable reaction product include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product. An improvement of the present method for determining the amount of hepcidin was achieved by including a subsequent step of incubating binding antibodies to allow binding to the hepcidin bound to the chicken antibodies. In such a sandwich ELISA the antibodies used for incubation in the subsequent step are rabbit polyclonal antibodies. Thus in a further preferred embodiment of the present invention, the method comprises after step b) the step of contacting the incubated chicken antibodies with polyclonal antibodies from rabbit immunized with hepcidin that allow binding of polyclonal rabbit antibodies to the hepcidin bound to the chicken antibodies.
The present invention thus also concerns a method for determining the amount of hepcidin in a sample comprising a) obtaining a sample to be assayed for human hepcidin, bl) incubating the sample with polyclonal antibodies from chicken immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample, b2) contacting the incubated chicken antibodies with polyclonal antibodies from rabbit immunized with hepcidin that allow binding of polyclonal rabbit antibodies to the hepcidin bound to the chicken antibodies and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay.
The hepcidin that is used for immunisation of rabbits is preferably the same as that is used for immunization of chicken which preferably is human hepcidin-25. The human hepcidin-25 that is used for immunization can be prepared by any method such as via recombinant expression technology or via synthesis. In a suitable immunization protocol rabbits at start immunization are immunized with synthetic hepcidin, emulsified in an equal volume of Freund's complete adjuvant. Injections can be subcutaneously, but advantageously the first injection is in the popliteal gland. Booster inoculations with a similar amount of hepcidin emulsified in Freund's incomplete adjuvant are administered subcutaneously. Injections may be repeated every two weeks but advantageously injections are repeated at 4-week intervals. Injections are repeated for example 5-15 times, e.g. about 10 times. Just before each injection blood, about 5 or 6 ml, is collected. A suitable amount of hepcidin to use at start immunization is about 10-30 μg in 250-500 μΐ PBS.
Polyclonal antibodies against hepcidin were purified by affinity chromatography. Suitably rabbit anti-hepcidin IgG is purified by affinity chromatography on hepcidin-24 coupled columns.
In the sandwich-type assay involving chicken- and rabbit-anti hepcidin antibodies, binding of the rabbit antibodies is preferably carried out in a non-competitive assay. Thus preferably in step c) the amount of rabbit antibody that is bound to hepcidin that is bound by chicken antibody is measured.
For detection purposes, the rabbit anti-hepcidin antibodies may either be labelled or unlabeled. Advantageously, the rabbit anti-hepcidin antibodies may be determined by a label that can be indirectly detected by the production of a detectable reaction product including various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product.
A preferred method for carrying out the immunoassay according to the invention involves immobilising the chicken polyclonal antibody on a solid support. When this immobilisation is carried out directly onto the solid support itself, e.g. a microtiter plate, such immobilisation may lead to conformational changes of the antibody and may also lead to less reproducibility. Therefore in a preferred embodiment, the present method comprises binding the chicken polyclonal antibody to anti-chicken antibody which is immobilized on a solid support. Advantageously the anti-chicken antibody is duck anti-chicken antibody which minimises disturbances. Duck anti-chicken antibody can be prepared as described in Grebenchtchikov et al. (2002), 268:219-231. In a further embodiment, to not have to interfere with the isolated polyclonal anti-hepcidin rabbit antibodies, it is preferred not to label the rabbit antibodies. Preferably the rabbit antibodies are detected by anti-rabbit antibody. Preferably the anti-rabbit antibody is labelled. Preferably the anti-rabbit antibody is goat anti-rabbit antibody. Advantageously, the anti-rabbit antibody may be determined by a label that can be indirectly detected by the production of a detectable reaction product including various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, [beta]-galactosidase, xanthine oxidase, glucose oxidase or other saccharide oxidases, or luciferases, which transform appropriate substrate to form a coloured or fluorescent reaction product. Preferably the label is horseradish peroxidase. The order of incubating with polyclonal antibodies in the sandwich-type ELISA may be altered, thus steps bl) and b2) may be switched. Thus in one embodimentcomprising the present method comprises a) obtaining a sample to be assayed for human hepcidin, bl) incubating the sample with polyclonal antibodies from rabbit immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample, b2) contacting the incubated rabbit antibodies with polyclonal antibodies from chicken immunized with hepcidin that allow binding of polyclonal chicken antibodies to the hepcidin bound to the rabbit antibodies and c) determining the amount of bound antibodies or bound hepcidin by an immunodetection method and preferably the immunodetection method is an enzyme-linked immunosorbent assay. In one embodiment the present method is for determining the amount of human hepcidin-25. In another embodiment the present method is for determining the amount of hepcidin highly homologous to human hepcidin-25, in particular for Cynomologus Monkey hepcidin.
In another embodiment the invention relates to a kit of parts for carrying out the method according to the present invention comprising anti hepcidin polyclonal chicken antibody, and preferably also anti hepcidin polyclonal rabbit antibody, and preferably also duck anti-chicken antibody and labeled goat anti-rabbit antibody or labeled duck anti-chicken antibody and goat anti-rabbit antibody. Preferably the kit of parts comprises anti hepcidin polyclonal chicken antibody, anti hepcidin polyclonal rabbit antibody, duck anti-chicken antibody and labelled goat anti-rabbit antibody.
Examples
Immunisation of chicken
Chickens were initially immunized intramuscularly (pectoral) with synthetic hepcidin -25 (10-15 μg in 250 μΐ PBS), emulsified with an equal volume of Freund's complete adjuvant. Booster inoculations, containing 10-15 μg immunogen prepared with Freund's incomplete adjuvant, were administered every two weeks. Approximately 42 days after start immunization eggs from the immunized chickens were collected daily (collection).
Isolation of IgY fraction from the egg yolk and affinity purification of the polyclonal antibodies were performed as previously described(Polsen et al. Immunol Invest (1985), 14:323-327.): After separation of egg-yolk from egg-white and intensive flushing with water, the yolk membrane was pierced and the yolk content was collected in a tube. The volume was adjusted to 30 ml with PBS and the solution obtained was chilled on ice. Subsequently 30 ml of 7% PEG in PBS (final concentration 3.5%) was added and the mixture was left for 10 min at 4°C. The mixture was then centrifuged at 5,000xg for 20 min at 4°C. To the supernatant (ca. 40 ml), which was filtered through a hydrophilic gauze and chilled on ice, 10 ml of 50% PEG in PBS (final concentration 10%) was added and the mixture was left for 10 min at 4°C. The mixture was then centrifuged for 25 minutes at 5,000xg at 4°C. The pellet was suspended in 20 ml of PBS, chilled on ice, and 20 ml of 24% PEG in PBS (final concentration 12%) was slowly added. After 10 minutes the suspension was centrifuged at 5,000xg during 25 min at 4°C, the supernatant discarded and the pellet was resuspended in 4 ml of PBS and chilled on ice. To the suspension obtained 4 ml of ice-cold (-20°C) 50% ethanol and after 10 minutes, the suspension was centrifuged at 10,000xg for 25 min at 4°C and the pellet was resuspended in 4 ml of PBS. The recovery of IgY during this procedure was found to be ca. 50% and the purity of the isolates was estimated as approximately 90% (SDS-PAGE). Isolates from 8-10 eggs were pooled and divided into aliquots equal to the number of eggs used. The temperature of isolates storage was -20°C. Polyclonal anitbodies against hepcidin were purified by affinity chromatography on hepcidin-24 coupled columns (Affi-Gel® 10/15; Bio-Rad Laboratories, Hercules, CA, USA). Before being applied to the columns the samples of antibodies (ca. 4 ml) were filtrated and thereafter passed through the columns three times. The elution of antibodies from the columns was performed after the washing with 50 ml of PBS. The chicken antibodies were eluted with 8 ml of 5.0 mol/L KSCN in 50% ethylene glycol/water. Antibodies were stored in aliquots at -20°C
Immunisation of rabbits
Synthetic human hepcidin was used for immunizing rabbits. Aliquots containing 10 μg hepcidin- 25 in 500 μΐ PBS were mixed with an equal volume of Freund's complete adjuvant for the first injection and with Freund's incomplete adjuvant for boosters. The first injection was in the popliteal gland, whereas booster inoculations were administered subcutaneously. Injections were repeated nine times at 4-weekintervals. Blood (5 ml) was collected just before the injections in citrate tubes. After centrifugation the citrate plasma was stored at -20°C.
Polyclonal antibodies against hepcidin were purified by affinity chromatography on hepcidin-24 coupled columns (Affi-Gel® 10/15; Bio-Rad Laboratories, Hercules, CA, USA). Before being applied to the columns the samples of antibodies (ca. 4 ml) were filtrated and thereafter passed through the columns three times. The elution of antibodies from the columns was performed after the washing with 50 ml of PBS. The rabbit antibodies were eluted with 8 ml of PBS containing 1.25 mol/L KSCN. The purified rabbit anti-hepcidin antibodies were concentrated approximately 10 times by Amicon Ultra- 15 centrifugal filter device (Millipore, Amsterdam, the Netherlands) and, after dilution with glycerol (1 :1), stored at -20°C.
Competitive ELISA
Hepcidin-25 was biotinylated by incubating 1 1 .5 μg hepcidin-25 (Peptide International, Louisville, KY, USA) with 8.17 μg No-Weigh™ Sulfo-NHS-Biotin (20-fold molar excess) (Pierce, Rockford, IL, USA) during 30 minutes at ambient temperature. The procedure started with coating of the microtiter plate (96-well Nunc Maxisorb™ flat bottomed) with 100 μίΛνεΙΙ of duck-anti-chicken IgY antibody (Grebenchtchikov et al. (2002), 268:219-231) (4 mg/L in 50 mmol/L NaHC03/Na2C03, pH 9.6) overnight at 4°C. The plate was then washed four times with 300 μίΛνεΙΙ of washing buffer (1 mL/L Tween-20 in PBS) by use of a 96PW plate washer (Tecan, Mannedorf, Switzerland). The plates were blocked with 10 g L BSA (Sigma Aldrich, St. Louis, MO, USA) in PBS (300 μΐ ννεΐΐ) for 2h at ambient temperature and washed four times with washing buffer. The next step was the incubation with chicken-anti-hepcidin antibody [100 μΙ^ΛνεΙΙ; 0.3 mg/L in dilution buffer (10 g/L BSA in washing buffer)] for 2h at ambient temperature. After four washings, the calibrator (hepcidin-25), the unknown samples (20-fold diluted in dilution buffer unless stated otherwise), and the reference sample (75 μΐ ννεΐΐ in dilution buffer) were added to the wells, whereafter 75 μΐ ννεΐΐ biotinylated hepcidin (dilution 10,000x) in dilution buffer was added and the plates were incubated overnight at 4°C. The plates were washed four times with 300 μΐ washing buffer and subsequently plates were incubated with 100 μΕ/well Streptavidin-POD conjugate (peroxidase conjugated to streptavidin; Roche Diagnostics GmbH, Penzberg, Germany) (dilution 50,000x) during lh at ambient temperature. Plates were washed 5 times with 300 μΐ washing buffer and incubation with substrate solution (4 mg OPD in borate/citrate buffer) was performed in darkness for 15 minutes. The color reaction was stopped by addition of 100 μΐ H2SO4 per well and optical density was measured using an automated ELISA reader (Lab Systems, Oy, Helsinki, Finland) at 492 nm.
Results:
Use of chicken hepcidin AB's in a one-site ELISA yielded an unrecorded analytical sensitivity of 0.02 ng/ml, which is important for the proper diagnosis of iron disorders that are accompanied by very low circulating hepcidin levels that are currently undetectable.
Sensitive 2 -site ELISA
An enzyme-linked immunosorbent assay (ELISA) for human hepcidin was developed according to the 4-span approach.
The sandwich structure employed includes four different antibodies (Abs), a coating Ab (duck anti-chicken IgY) a capture Ab (chicken anti-hepcidin), a trapping Ab (rabbit anti-hepcidin) and finally a detection Ab (HRP-labeled goat anti-rabbit). The assay is insensitive to heterophilic antibodies. All incubation steps were separated by washing steps (4 times, 300 μΐ of washing buffer per well). The coating Ab was diluted in the coating buffer, whereas capture, trapping and detecting Abs as well as standards, unknowns and reference sample were diluted in the dilution buffer (100 μΐ per well). The microtiter plates were first overnight coated at 4°C with coating antibody (duck anti-chicken IgY) antibody, and then treated for 2 hours at 37°C with blocking buffer. Next, the plate was incubated for 2 hours at RT with capture antibody (chicken anti-hepcidin) and the samples, reference samples and standards were incubated overnight at 4°C. Trapping antibody (rabbit anti- hepcidin) and detection antibody (HRP-labeled goat anti-rabbit, commercially available from Thermo Fisher Scientific, Pierce Protein Research Products, Rockford, IL, USA) were sequentially incubated for 2 hours at RT and plates were developed with substrate solution in the dark for 30 min at RT, color reaction was stopped with H2SO4, and optical density was measured at 492 nm within 30 minutes.
Results:
Use of chicken hepcidin AB's followed by rabbit hepcidin AB's in a two-site ELISA yielded a further improved analytical sensitivity of 0.006 ng/ml.

Claims

Claims
1. A method for determining the amount of hepcidin in a sample comprising a) obtaining a sample to be assayed for human hepcidin, b) incubating the sample with polyclonal antibodies from chicken immunized with hepcidin under conditions that allow binding of the polyclonal antibodies to the human hepcidin in the sample and c) determining the amount of bound antibodies or bound hepcidin by an enzyme-linked immunosorbent assay (ELISA).
2. The method according to claim 1 wherein the enzyme-linked immunosorbent assay is a competitive-ELISA.
3. The method according to claim 1 , comprising after step b) contacting the incubated chicken antibodies with polyclonal antibodies from rabbit immunized with hepcidin that allow binding of polyclonal rabbit antibodies to the hepcidin bound to the chicken antibodies.
4. The method according to claim 3 wherein the enzyme-linked immunosorbent assay is a non- competitive-ELISA.
5. The method according any one of the preceding claims, wherein the chicken and rabbit are immunized with human hepcidin-25.
6. The method according any one of the preceding claims, comprising binding the chicken polyclonal antibody to anti-chicken antibody which is immobilized on a solid support.
7. The method according to claim 6, wherein the anti-chicken antibody is duck anti-chicken antibody.
8. The method according any one of claims 3-7, wherein in step c) is incubated with labeled anti-rabbit antibody.
9. The method according to claim 8, wherein the labeled anti-rabbit antibody is goat labeled anti-rabbit antibody.
10. The method according to claim 8 or 9 wherein the label is horseradish peroxidase.
1 1. The method according any one of the preceding claims, wherein the sample is isolated from a human.
12. The method according to any of the preceding claims wherein the sample is isolated or derived from plasma, serum or urine of a human.
13. Kit of parts for carrying out the method according to any one of the preceding claims, comprising anti hepcidin polyclonal chicken antibody, anti hepcidin polyclonal rabbit antibody, duck anti-chicken antibody and labeled goat anti-rabbit antibody.
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