Immunoassay tracer reagents and methods for measuring the amount of native human bioactive hepcidin

Abstract

The invention provides compositions and methods for measuring human serum hepcidin levels. The invention provides methods for the oxidative refolding of a hepcidin polypeptide to a form that is mature, bioactive and folded as in the native configuration and molecular mass; a method for measuring the level of native, bioactive hepcidin in a vertebrate animal.

1 - 18 . (canceled) 
     
     
         19 : An immunoassay tracer reagent comprising:
 (a) (i) an oxidatively folded hepcidin peptide consisting of SEQ ID NO:28;   (ii) a hydrophilic spacer comprising one or more (2-(2-Amino-Ethoxy)Ethoxy) Acetic Acid (AEEAc) residues, wherein the peptide of (i) is covalently linked to the hydrophilic spacer at its amino terminus, its lysine at position 18, or lysine at position 24; and   (iii) a detectable marker covalently linked to the spacer residue of (ii).   
     
     
         20 : The immunoassay tracer reagent of claim  37 , wherein
 (i) the detectable marker comprises a biotin, a fluorescent molecule or an enzyme; or   (ii) the detectable marker comprises a horseradish peroxidase (HRP) enzyme.   
     
     
         21 : The immunoassay tracer reagent of claim  37 , wherein the hydrophilic spacer comprises two (2-(2-Amino-Ethoxy)Ethoxy)Acetic Acid (AEEAc) residues. 
     
     
         22 : The immunoassay tracer reagent of claim  37 , wherein the hydrophilic spacer consists of two (2-(2-Amino-Ethoxy)Ethoxy)Acetic Acid (AEEAc) residues. 
     
     
         23 : The immunoassay tracer reagent of claim  37 , wherein the hepcidin peptide comprises an isolated, synthetic or recombinant peptide. 
     
     
         24 : The immunoassay tracer reagent of claim  37 , wherein the hepcidin peptide is oxidatively folded by a process comprising:
 (a) solubilizing the hepcidin peptide consisting of SEQ ID NO:28 in an acetic acid solution to produce a first solution;   (b) diluting the first solution with an aqueous buffer solution containing a chaotropic reagent, an organic alcohol, and an oxidizing reagent to produce a second solution; and   (c) adjusting the pH of the second solution to a level between approximately 5 and 7.   
     
     
         25 : A method for measuring the amount of native human bioactive hepcidin in a sample of tissue or bodily fluid taken from a human using a competitive Enzyme-Linked Immunosorbent Assay (ELISA) comprising:
 (a) obtaining a sample of a tissue or a bodily fluid from a human, and preparing a solution comprising the sample of the tissue or the bodily fluid;   (b) providing a known amount of the immunoassay tracer reagent of claim  37 ;   (c) providing an immobilized antibody that specifically binds to a bioactive human hepcidin, and contacting the immobilized antibody with a blocking solution;   (d) contacting a known amount of the solution of step (a) with a known amount of the reagent of step (b) in the presence of the immobilized antibody of step (c), thereby forming immobilized antibody-(human hepcidin) tracer reagent complexes and immobilized antibody-sample human hepcidin complexes;   (e) washing unbound tracer reagent from the reaction solution; and   (f) determining the amount of bioactive human hepcidin in the sample by competitive ELISA.   
     
     
         26 : A method for measuring the amount of bioactive human hepcidin in a sample of tissue or bodily fluid taken from a human using a competitive Enzyme-Linked Immunosorbent Assay (ELISA) comprising:
 (a) obtaining a sample of a tissue or a bodily fluid from a human, and preparing a solution comprising the sample of the tissue or the bodily fluid;   (b) providing a known amount of immunoassay tracer reagent of claim  37 ;   (c) providing an immobilized antibody that specifically binds to a bioactive human hepcidin, and contacting the immobilized antibody with a blocking solution;   (d) contacting a known amount of the solution of step (a) with a known amount of the reagent of step (b) in the presence of the immobilized antibody of step (c), thereby forming complexes of immobilized antibody and tracer reagent and immobilized antibody-sample human hepcidin complexes;   (e) washing unbound tracer reagent from the reaction solution;   (f) detecting the amount of tracer reagent of step (b) bound to the antibody of step (c); and   (g) calculating the amount of bioactive human hepcidin in the sample based on results of the detecting of step (f).