NOVEL LIPOCALIN-MUTEIN ASSAYS FOR MEASURING HEPCIDIN CONCENTRATION

1 - 36 . (canceled)

37 . A lipocalin-mutein assay, which comprises:

(i) a phase,

(ii) a lipocalin mutein or fragment or variant thereof that specifically binds to hepcidin, and

(iii) a control hepcidin or fragment or variant thereof;

wherein the control hepcidin or fragment or variant thereof competes with a non-control hepcidin in a biological sample for binding to the lipocalin mutein or fragment or variant thereof, when such sample is contacted with the assay; wherein either (ii) or (iii) serves as a tracer molecule that is captured on the phase by a capture reagent, which is the other one of (ii) or (iii); and wherein the tracer molecule can be detected and/or quantified via a label.

38 . The lipocalin-mutein assay of claim 37 , wherein the tracer molecule is at the concentration range of about 0.1 nM-3 nM.

39 . The lipocalin-mutein assay of claim 37 , which assay comprises a binding agent, wherein the control hepcidin or fragment or variant thereof is conjugated to a moiety and thereby can be captured by such binding agent.

40 . The lipocalin-mutein assay of claim 39 , which assay comprises a blocking agent.

41 . The lipocalin-mutein assay of claim 37 , wherein the mean value of the concentration of non-control hepcidins in a biological sample as measured by the lipocalin-mutein assay is within the same range of the mean value of the concentration of non-control hepcidins in a corresponding sample as measured by a mass spectrometry (MS) assay, which MS assay is essentially described in Murphy A T et al. Blood. 2007; 110:1048-1054.

42 . The lipocalin-mutein assay of claim 37 , wherein the lipocalin mutein or fragment or variant thereof is capable of binding hepcidin with an affinity measured by a KD of about 10 nM or lower.

43 . The lipocalin-mutein assay of claim 37 , wherein the lipocalin mutein is a hNGAL mutein, wherein the hNGAL mutein has at any two or more amino acids at a position corresponding to position 96, 100, and/or 106 of the linear polypeptide sequence of the mature hNGAL lipocalin a mutated amino acid, and wherein the hNGAL mutein further has one or more amino acids at a position corresponding to position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and/or 134 of the linear polypeptide sequence of mature hNGAL lipocalin (SEQ ID NO: 15) a mutated amino acid.

44 . The lipocalin-mutein assay of claim 43 , wherein the hNGAL mutein has the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, or a fragment or variant thereof.

45 . A method of preparing a lipocalin-mutein assay, comprising the steps of:

(i) immobilizing one or more lipocalin muteins or fragments or variants thereof on a phase, which lipocalin muteins or fragments or variants hereof specifically bind to hepcidin; and

(ii) providing one or more control hepcidins or fragments or variants at the concentration range of 0.1 nM-3 nM.

46 . The method of claim 45 , further comprising the step of adding a blocking agent after step (i).

47 . A method of preparing a lipocalin-mutein assay, comprising the steps of:

(i) immobilizing one or more binding agents on a phase;

(ii) providing one or more control hepcidins or fragments or variants thereof, which control hepcidins or fragments or variants thereof are conjugated to a moiety and thereby can be captured by the binding agents; and

(iii) providing lipocalin muteins or fragments or variants thereof at the concentration range of 0.1 nM-3 nM, which lipocalin muteins or fragments or variants hereof specifically bind to hepcidin.

48 . The method of claim 47 , further comprising the step of adding a blocking agent after step (i).

49 . A method for quantitatively measuring a biological sample's hepcidin concentration, comprising the steps of:

(i) contacting a biological sample obtained from a subject with the lipocalin-mutein assay of claim 37 ;

(ii) measuring the signal level generated by one or more tracer molecules, captured on the phase, via one or more labels and/or a suitable instrument for signal detection; and

(iii) correlating the signal level on a standard curve with the biological sample's hepcidin concentration.

50 . The method of claim 49 , further comprising the steps of:

(iv) contacting various known concentrations of non-control hepcidins with the lipocalin-mutein assay; and

(v) measuring the signal levels corresponding to the various concentrations of step (iv) to generate a standard curve, which signal levels are generated by one or more tracer molecules, captured on the phase, via one or more labels and/or a suitable instrument for signal detection.

51 . The method of claim 50 , wherein the steps (iv) and (v) are carried out simultaneously with steps (i) and (ii), respectively.

52 . A method for identifying an altered level of hepcidin concentration in a subject, comprising:

(i) quantitatively measuring a biological sample's hepcidin concentration using a method according to claim 49 , wherein the biological sample is obtained from the subject; and

(ii) comparing the hepcidin concentration measured in step (i) with the prior-measured hepcidin concentration(s) of one or more corresponding sample(s) obtained from the subject.

53 . A method for identifying an altered level of hepcidin concentration in a subject, comprising:

(i) quantitatively measuring a biological sample's hepcidin concentration using the method according to claim 49 , wherein the biological sample is obtained from the subject; and

(ii) quantitatively measuring hepcidin concentration(s) of one or more corresponding sample(s) using the method according to claim 49 , wherein the corresponding sample(s) are obtained from the subject; and

(iii) comparing the hepcidin concentration measured in step (i) with the hepcidin concentration(s) of the one or more corresponding sample(s) measured in step (ii).

54 . A method for diagnosing a disease or disorder characterized by a non-physiological concentration of hepcidin in a subject, comprising:

(i) quantitatively determining a biological sample's hepcidin concentration according to claim 49 , wherein the biological sample is obtained from the subject; and

(ii) analyzing whether the hepcidin concentration measured in step (i) is non-physiological,

wherein the non-physiological concentration of hepcidin is an indicative of the disease or disorder in the subject.

55 . The method of claim 54 , wherein the analysis in step (ii) including comparing the hepcidin concentration measured in step (i) with the hepcidin concentration of a control sample, which is known to possess a normal hepcidin concentration.