Method for diagnosing or monitoring kidney function or diagnosing kidney dysfunction

Abstract

A method for (a) diagnosing or monitoring kidney function in subject or (b) diagnosing kidney dysfunction in a subject or (c) predicting or monitoring the risk of an adverse events in a diseased subject or (d) predicting or monitoring the success of a therapy or intervention comprising determining the level of Pro-Enkephalin (PENK) or fragments thereof of at least 5 amino acids in a bodily fluid obtained from said subject; and correlating said level of Pro-Enkephalin or fragments thereof with (a) kidney function in a subject or (b) kidney dysfunction in said subject or (c) enhanced risk of adverse events or (d) success of a therapy or intervention in a diseased subject.

Claims

1. A method for (a) diagnosing or monitoring kidney function in a subject or (b) diagnosing kidney dysfunction in a subject or (c) predicting or monitoring the risk of an adverse event in a subject with kidney disease wherein said adverse event is selected from the group comprising worsening of kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or death due to kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or (d) predicting or monitoring the success of a therapy or intervention in a subject with kidney disease comprising: determining the level of Pro-Enkephalin or fragments thereof in a bodily fluid obtained from said subject; and (a) correlating said level of Pro-Enkephalin or fragments thereof with kidney function in a subject, or (b) correlating said level of Pro-Enkephalin or fragments thereof with kidney dysfunction, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or (c) correlating said level of Pro-Enkephalin or fragments thereof with said risk of an adverse event in a subject with a kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or (d) correlating said level of Pro-Enkephalin or fragments thereof with success of a therapy or intervention in a subject with a kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 1 or SEQ ID No. 6, wherein the level of Pro-Enkephalin or fragments thereof is determined by using an assay comprising two binders that bind to two different regions within the region of Pro-Enkephalin that is amino acid 133-140 (LKELLETG, SEQ ID NO. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each of said regions comprises at least 4 amino acids.

2. A method according to claim 1 wherein the binders are antibodies, antibody fragments or a non-Ig-Scaffold binding to Pro-Enkephalin or fragments thereof.

3. A method according to claim 1 comprising determining the level of immunoreactive analyte by using an assay comprising two binders that bind to two different regions within the region of Pro-Enkephalin that is amino acid 133-140 (LKELLETG, SEQ ID NO. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each of said regions comprises at least 4 amino acids; and (a) correlating said level of immunoreactive analyte with kidney function in a subject, or (b) correlating said level of immunoreactive analyte with kidney dysfunction wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or (c) correlating said level of immunoreactive analyte with said risk of an adverse event in a subject with a kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or (d) correlating said level of immunoreactive analyte with success of a therapy or intervention in a subject with a kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 1 or SEQ ID No. 6.

4. A method according to claim 1, wherein said binders bind to a region within an amino acid sequence which is one or more of SEQ ID No. 1 or 6.

5. A method according to claim 1, wherein said threshold level of Pro-Enkephalin or fragments thereof is 80 pmol/L.

6. A method according to claim 1, wherein the level of Pro-Enkephalin is measured with an immunoassay and each of said binders is an antibody, or an antibody fragment or a non-Ig-Scaffold binding to Pro-Enkephalin or fragments thereof.

7. A method according to claim 1, wherein an assay is used for determining the level of Pro-Enkephalin or fragments thereof and wherein the assay sensitivity of said assay is able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is <15 pmol/L.

8. A method according to claim 1, wherein said bodily fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal liquid (csf), and saliva.

9. A method according to claim 1, wherein at least one additional clinical parameter is determined selected from the group comprising: age, BUN, NGAL, Creatinine Clearance, Creatinine and Apache Score.

10. A method according to claim 1, wherein said determination of Pro-Enkephalin or fragments thereof is performed more than once in one subject.

11. A method according to claim 1, wherein said monitoring is performed in order to evaluate the response of said subject to preventive and/or therapeutic measures taken.

12. A method according to claim 1, in order to stratify said subjects into risk groups.

13. A method according to claim 1, wherein said level of Pro-Enkephalin or fragments thereof are correlated with a risk of death or an adverse event in a subject with a kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of death or adverse events and wherein said subject with a kidney disease is male.

14. A point-of-care device for performing assays near a subject, wherein said point-of-care device is capable of performing a method of claim 1 and comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID NO. 14).

15. A kit which comprises: at least two antibodies or antibody fragments directed to either amino acid 133-140 (LKELLETG, SEQ ID No. 13) or amino acid 152-159 (SDNEEEVS, SEQ ID NO. 14).

16. A method according to claim 1, wherein said binders do not bind to enkephalin peptides [Met]enkephalin SEQ ID No:3, and [Leu]enkephalin. SEQ ID No: 4.

17. A method according to claim 1, wherein said binders bind to SEQ ID No. 6.

18. A method according to claim 1, wherein said binders bind to a region within an amino acid sequence SEQ ID No. 6.

19. A method comprising determining the level of Pro-Enkephalin fragments in a bodily fluid obtained from a subject using a binder to said Pro-Enkephalin fragments; and correlating said level of said Pro-Enkephalin fragments with kidney function in said subject, or kidney dysfunction in said subject, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin fragments is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11.

20. A method according to claim 19 additionally comprising determining the level of immunoreactive analyte by using at least one binder that binds to a region within the amino acid sequence of Pro-Enkephalin fragments in a bodily fluid obtained from said subject; and correlating said level of immunoreactive analyte with kidney function in said subject, or kidney dysfunction in said subject, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in said subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin fragment is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11.

21. A method for (a) diagnosing or monitoring kidney function in a subject or (b) diagnosing kidney dysfunction in a subject or (c) predicting or monitoring the risk of an adverse events in a subject, wherein said adverse event is selected from the group comprising worsening of kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or death due to kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or (d) predicting or monitoring the success of a therapy or intervention comprising determining the level of Pro-Enkephalin or fragments thereof in a bodily fluid obtained from said subject; and (a) correlating said level of Pro-Enkephalin or fragments thereof with kidney function in said subject, or (b) correlating said level of Pro-Enkephalin or fragments thereof with kidney dysfunction, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or (c) correlating said level of Pro-Enkephalin or fragments thereof with said risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or (d) correlating said level of Pro-Enkephalin or fragments thereof with success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragments thereof is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11, wherein the level of Pro-Enkephalin or fragments thereof is determined by using an assay comprising a binder, wherein the binder is selected from the group consisting of an antibody, an antibody fragment, or a non-Ig-Scaffold that does not bind to enkephalin peptides [Met]enkephalin SEQ ID No:3.

22. A method according to claim 21 wherein the binder is selected from the group consisting of an antibody, an antibody fragment, or a non-Ig-Scaffold binding to Pro-Enkephalin or fragments thereof that also does not bind to [Lu]enkephalin. SEQ ID No: 4.

23. A method according to claim 21 further comprising determining the level of immunoreactive analyte in a bodily fluid obtained from said subject by using an assay comprising a binder that binds to a region within the amino acid sequence of Pro-Enkephalin (PENK) or fragments thereof but does not bind to enkephalin peptides [Met]enkephalin SEQ ID No: 3; and correlating said level of immunoreactive analyte with kidney function in a subject, or kidney dysfunction, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse event, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11.

24. A method according to claim 23 wherein the binder is selected from the group consisting of an antibody, an antibody fragment, or a non-Ig-Scaffold binding to Pro-Enkephalin or fragments thereof that also does not bind to [Leu]enkephalin SEQ ID No: 4.

25. A method according to claim 21, wherein said threshold level of Pro-Enkephalin or fragments thereof is 80 pmol/L.

26. A method according to claim 21, wherein an assay is used for determining the level of Pro-Enkephalin or fragments thereof and wherein the assay sensitivity of said assay is able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is <15 pmol/L.

27. A point-of-care device for performing assays near a subject, wherein said point-of-care device is capable of performing a method of claim 21 and comprises an antibody or antibody fragment that does not bind to enkephalin peptides [Met]enkephalin SEQ ID No: 3.

28. A kit which comprises: a) a point-of-care device for performing assays near a subject, capable of performing a method of claim 21 and b) an antibody or antibody fragment that does not bind to enkephalin peptides [Met]enkephalin SEQ ID No: 3.

29. A method for (a) diagnosing or monitoring kidney function in subject or (b) diagnosing kidney dysfunction in a subject or (c) predicting or monitoring the risk of an adverse events in a subject wherein said adverse event is selected from the group comprising worsening of kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or death due to kidney dysfunction including kidney failure, loss of kidney function and end-stage kidney disease or (d) predicting or monitoring the success of a therapy or intervention comprising determining the level of Pro-Enkephalin or fragments thereof in a bodily fluid obtained from said subject; and (a) correlating said level of Pro-Enkephalin or fragments thereof with kidney function in a subject, or (b) correlating said level of Pro-Enkephalin or fragments thereof with kidney dysfunction, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or (c) correlating said level of Pro-Enkephalin or fragments thereof with said risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events, or (d) correlating said level of Pro-Enkephalin or fragments thereof with success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11, wherein the level of Pro-Enkephalin or fragments thereof is determined by using an assay with an assay sensitivity able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is <15 pmol/L.

30. A method according to claim 29 additionally comprising determining the level of immunoreactive analyte in a bodily fluid obtained from said subject by using an assay with at least one binder that binds to a region within the amino acid sequence of Pro-Enkephalin (PENK) or fragments thereof with an assay sensitivity able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is <15 pmol/L; and correlating said level of immunoreactive analyte with: kidney function in a subject, or kidney dysfunction, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse event, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11.

31. A method for diagnosing or monitoring kidney function using a kit or assay to detect Pro-Enkephalin or fragments thereof in a subject, wherein said kit or assay comprises at least one antibody capable of binding Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said subject, said method comprising: determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from a subject using an assay comprising an antibody that binds to Pro-Enkephalin or fragments thereof; wherein said Pro-Enkephalin or fragment is one or more of SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 or SEQ ID No. 11 correlating said level of Pro-Enkephalin or fragments thereof with kidney function in a subject, or kidney dysfunction wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse event, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention.

32. A method of using a kit or assay according to claim 31 additionally comprising determining the level of immunoreactive analyte by using an assay comprising an antibody that binds to a region within the amino acid sequence of Pro-Enkephalin or fragments thereof in a bodily fluid obtained from said subject; and correlating said level of immunoreactive analyte with kidney function in a subject, or kidney dysfunction in a subject, wherein an elevated level above a certain threshold is predictive or diagnostic for kidney dysfunction in said subject, or risk of an adverse event in a subject with kidney disease, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse event, or success of a therapy or intervention in a subject with kidney disease, wherein a level below a certain threshold is predictive for a success of therapy or intervention, wherein said Pro-Enkephalin or fragment is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11.

33. A method according to claim 31, wherein the antibody of said kit or assay does not bind to enkephalin peptides [Me]enkephalin SEQ ID No: 3, and [Leu]enkephalin. SEQ ID No: 4.

34. A method according to claim 31, wherein the antibody of said kit or assay binds to SEQ ID No. 6.

35. A method according to claim 31, wherein the antibody of said kit or assay binds to a region within an amino acid sequence selected from the group consisting of SEQ ID No. 1, 2, 5, 6, 7, 8, 9 and 10.

36. A method according to claim 31, wherein the antibody of said kit or assay binds to a region within an amino acid sequence selected from the group consisting of SEQ ID No. 2, 5, 6, and 10.

37. A method according to claim 31, wherein the sensitivity of the assay used for determining the level of Pro-Enkephalin or fragments thereof is able to quantify the Pro-Enkephalin or Pro-Enkephalin fragments of healthy subjects and is <15 pmol/L.

Description

EXAMPLES

Example 1

(1) Development of Antibodies

(2) Peptides

(3) Peptides were synthesized (JPT Technologies, Berlin, Germany).

(4) Peptides/Conjugates for Immunization:

(5) Peptides for immunization were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal Cystein residue for conjugation of the peptides to bovine serum albumin (BSA). The peptides were covalently linked to BSA by using Sulfo-SMCC (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

(6) TABLE-US-00003 TABLE1 Peptidefor Pro-Enkephalin- immunization sequence (C)DAEEDD 119-125 (C)EEDDSLANSSDLLK 121-134 (C)LKELLETG 133-140 (C)TGDNRERSHHQDGSDNE 139-155 (C)SDNEEEVS 152-159

(7) The antibodies were generated according to the following method:

(8) A BALB/c mouse was immunized with 100 g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 l complete Freund's adjuvant) and 50 g at day 21 and 28 (in 100 l incomplete Freund's adjuvant). Three days before the fusion experiment was performed, the animal received 50 g of the conjugate dissolved in 100 l saline, given as one intraperitonal and one intravenous injection.

(9) Spenocytes from the immunized mouse and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37 C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-supplement]. After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

(10) The cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion. The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and recloned using the limiting-dilution technique and the isotypes were determined.

(11) (Lane, R. D. A short-duration polyethylene glycol fusiontechnique for increasing production of monoclonal antibody-secreting hybridomas, J. Immunol. Meth. 81: 223-228; (1985), Ziegler, B. et al. Glutamate decarboxylase (GAD) is not detectable on the surface of rat islet cells examined by cytofluorometry and complement-dependent antibody-mediated cytotoxicity of monoclonal GAD antibodies, Horm. Metab. Res. 28: 11-15, (1996)).

(12) Monoclonal Antibody Production

(13) Antibodies were produced via standard antibody production methods (Marx et al., Monoclonal Antibody Production (1997), ATLA 25, 121) and purified via Protein A-chromatography. The antibody purities were >95% based on SDS gel electrophoresis analysis.

(14) Labelling and Coating of Antibodies.

(15) All antibodies were labelled with acridinium ester according the following procedure:

(16) Labelled compound (tracer): 100 g (100 l) antibody (1 mg/ml in PBS, pH 7.4), was mixed with 10 l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature. Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/1 potassiumphosphate, 100 mmol/1 NaCl, 10 mmol/1 Na-EDTA, 5 g/1 bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).

(17) Solid phase antibody (coated antibody):

(18) Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 g antibody/0.3 ml 100 mmol/1 NaCl, 50 mmol/l Tris/HCl, pH 7.8). After blocking with 5% bovine serum albumine, the tubes were washed with PBS, pH 7.4 and vacuum dried.

(19) Antibody Specificity

(20) TABLE-US-00004 TABLE2 Peptidefor Pre-Pro-Enkephalin- Antibody immunization sequence name (C)DAEEDD 119-125 NT-MRPENK (C)EEDDSLANSSDLLK 121-134 NM-MRPENK (C)LKELLETG 133-140 MR-MRPENK (C)TGDNRERSHHQDGSDNE 139-155 MC-MRPENK (C)SDNEEEVS 152-159 CT-MRPENK

(21) Antibody cross-reactivities were determined as follows:

(22) lug peptide in 300 l PBS, pH 7,4 was pipetted into Polystyrene tubes and incubated for 1 h at room temperature. After incubation the tubes were washed 5 times (each 1m1) using 5% BSA in PBS, pH 7.4. Each of the labelled antibodies were added (300 l in PBS, pH 7.4, 800.000 RLU/300 l) an incubated for 2 h at room temperature, After washing 5 times (each 1 ml of washing solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X 100), the remaining luminescence (labelled antibody) was quantified using the AutoLumat Luminumeter 953. MRPENK-peptide was used as reference substance (100%).

(23) The crossreactivities of the different antibodies are listed in table 3.

(24) TABLE-US-00005 TABLE3 MRPENK (SEQ Anti- DAEE EEDDSLANSS LKELLE TGDNRERSHH SDNEEE ID body DD DLLK TG QDGSDNE VS NO.6) NT- 121 10 <1 <1 <1 100 MRPENK NM- <1 98 <1 <1 <1 100 MRPENK MR- <1 <1 105 <1 <1 100 MRPENK MC- <1 <1 <1 115 <1 100 MRPENK CT- <1 <1 <1 <1 95 100 MRPENK

(25) All antibodies bound the MRPENK peptide, comparable to the peptides which were used for immunization. Except for NT-MRPENK-antibody (10% cross reaction with EEDDSLANSSDLLK), no antibody showed a cross reaction with MR-PENK fragments not used for immunization of the individual antibody.

(26) Pro-Enkephalin Immunoassay:

(27) 50 l of sample (or calibrator) was pipetted into coated tubes, after adding labeled antibody (200 l), the tubes were incubated for 2 h at 18-25 C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mmol/l PBS, pH 7.4, 0.1% Triton X-100). Tube-bound labelled antibody was measured by using the Luminumeter 953. Using a fixed concentration of 1000 pmol/of MRPENK. The signal (RLU at 1000 pmol MRPENK/1) to noise (RLU without MRPENK) ratio of different antibody combinations is given in table 4. All antibodies were able to generate a sandwich complex with any other antibody. Surprisingly, the strongest signal to noise ratio (best sensitivity) was generated by combining the MR-MRPENK- and CT-MRPENK antibody. Subsequently, we used this antibody combination to perform the MRPENK-immunoassay for further investigations. MR-MRPENK antibody was used as coated tube antibody and CT-MRPENK antibody was used as labelled antibody.

(28) TABLE-US-00006 TABLE4 Solid phase NT- NM- MR- MC- CT- antibody MRPENK MRPENK MRPENK MRPENK MRPENK Labelled antibody NT- / 27 212 232 <1 MRPENK NM- 36 / 451 487 <1 MRPENK MR- 175 306 / 536 1050 MRPENK MC- 329 577 542 / <1 MRPENK CT- <1 615 1117 516 / MRPENK
Calibration:

(29) The assay was calibrated, using dilutions of synthetic MRPENK, diluted in 20 mM K2PO4.6 mM EDTA, 0.5% BSA, 50 M Amastatin, 100 M Leupeptin, pH 8.0. Pro-Enkephalin control plasma is available at ICI-diagnostics, Berlin, Germany.

(30) FIG. 1 shows a typical Pro-Enkephalin dose/signal curve.

(31) The assay sensitivity was 20 determinations of 0-calibrator (no addition of MRPENK) +2SD) 5.5 pmol/L.

(32) Creatinine Clearance

(33) Creatinine clearance was determined using the MDRD formula (see Levey et al, 2009).

Example 2

(34) PENK in healthy subjects

(35) Healthy subjects (n=4211, average age 56 years) were measured using the MRPENK assay. The mean value was 44.7 pmol MRPENK/L, the lowest value was 9 pmol/L and the 99.sup.thpercentile was 80 pmol/L. Since the assay sensitivity was 5.5 pmol/L, 100% of all healthy subjects were detectable using the described MRPENK assay (see FIG. 2).

(36) Pro-Enkephalin correlates with Creatinine Clearance in healthy subjects with normal kidney function.

(37) Surprisingly, Pro-Enkephalin was negatively correlated with Creatinine Clearance in healthy subjects (r=0.33, p<0.0001), see FIG. 3. The coefficient of correlation was slightly stronger in male than in females (r=0.34 vs 0.29, both p<0.0001). These data indicating a strong association between PENK and kidney function.

(38) FIG. 3: correlation of creatinine clearance vs. PENK in healthy subjects. Y axis: quartiles of Creatinine Clearance, x axis: quartiles of PENK.

Example 3

(39) Correlation of PENK and kidney function (creatinine clearance) in patients with chronic and acute diseases.

(40) TABLE-US-00007 TABLE 5 Disease r-value p-value Chronic Heart Failure 0.55 <0.0001 N = 122 Acute Heart Failure 0.68 <0.0001 N = 149 Acute Myocardial Infarction 0.82 <0.0001 N = 78 Sepsis 0.74 <0.0001 N = 101 SIRS 0.79 <0.0001 N = 109
PENK correlated always significantly with creatinine clearance, in acute diseases the correlation was stronger than in chronic diseases or in healthy subjects.

Example 4

PENK in critical ill patients

(41) To investigate the diagnostic performance of PENK for diagnosis of kidney failure in acute clinical settings, we performed the following clinical study:

(42) Clinical Study

(43) 101 ED patients fulfilling the definition of sepsis (Crit Care Med. 2008 Jan; 36(1):296-327.) were subsequently hospitalized (average 5 days of hospitalization) and received a standard of care treatment. EDTA-plasma was generated from day 1 (ED presentation) and one sample each day during hospital stay. The time to freeze samples for later analyte-measurement was less than 4 h.

(44) Patient characteristics are summarized in table 6:

(45) TABLE-US-00008 TABLE 6 all in hospital deaths discharged Variable (n = 101) (n = 27) (n = 74) p-value Demographics Gendermale 60 (60) 13 (48) 47 (64) 0.163 Agemedian [IQR] 78 [72-72] 77 [71.25-83] 80 [75-84.5] 0.142 Examination variables BP systolic (mmHg)median [IQR] 115 [100-100] 120 [106.25-138.75] 105 [80-120] 0.001 BP diastolic (mmHg)median [IQR] 65 [60-60] 65 [60-85] 60 [50-70] 0.002 HRmedian [IQR] 100 [94-94] 100 [94-114.75] 100 [93.5-107.5] 0.407 RRmedian [IQR] 24 [22-22] 24 [22-28] 26 [24-28] 0.069 MAP (mmHg)median [IQR] 83.3 [74-74] 83.3 [77.62-100.75] 81.6 [63.5-89] 0.026 concomitant diseases Cardiovascularyes 26 (25.7) 9 (33.3) 17 (23) 0.311 Hypertensiveyes 47 (46.5) 13 (48.1) 34 (45.9) 1.000 Diabetesyes 35 (34.7) 9 (33.3) 26 (35.1) 1.000 Cancereyes 13 (12.9) 3 (11.1) 10 (13.5) 1.000 routine labaratory variables Blood cultureyes 31 (31) 5 (19) 26 (35) 0.246 negative 15 (16.3) 2 (8) 13 (19.4) positive 16 (17.4) 3 (12) 13 (19.4) Creatinine clearance (ml/min) 48 [23.25-23.25] 56 [29.25-80] 31.5 [14.75-66] 0.043 median [IQR] Creatininemedian [IQR] 1.3 [0.9-0.9] 1.25 [0.9-2.08] 1.8 [1-3.15] 0.080 UREAmedian [IQR] 36 [21-21] 31.5 [20-53.25] 51 [42-87] 0.004 GCSmedian [IQR] 15 [10-10] 15 [12.5-15] 8 [8-11] <0.001 Pcrmedian [IQR] 16 [6.6-6.6] 14.5 [6.7-23.7] 17.35 [6.6-28.05] 0.846 Glucomedian [IQR] 113.5 [94.5-94.5] 110 [95.5-144] 128 [94-160.5] 0.400 bilirumedian [IQR] 0.9 [0.71-0.71] 0.9 [0.7-1.03] 0.91 [0.77-1.18] 0.534 GRmedian [IQR] 3.8 [3.3-3.3] 3.8 [3.2-4.3] 3.7 [3.4-4.2] 0.684 GBmedian [IQR] 12700 [6774-6774] 13100 [8115-17565] 11920 [25.55-18790] 0.343 PLTmedian [IQR] 213 [150-150] 217 [154.75-301] 185 [130-236.5] 0.113 HCTmedian [IQR] 32 [28-28] 31.5 [28-37] 34 [31.25-39.5] 0.149 Leuco/Neutr (%)median [IQR] 87 [80-80] 86 [78.25-89.95] 91 [87-93.05] 0.001 HBmedian [IQR] 10.4 [9.47-9.47] 10.15 [9.3-12.4] 10.85 [9.9-12.67] 0.220 Namedian [IQR] 137 [134-134] 137 [133-141] 139 [134-144.5] 0.204 Kmedian [IQR] 3.9 [3.5-3.5] 3.9 [3.6-4.3] 3.9 [3.3-5.1] 0.982 INRmedian [IQR] 1.19 [1.1-1.1] 1.19 [1.1-1.4] 1.18 [1.04-1.36] 0.731 TCmedian [IQR] 38.4 [36-36] 38.5 [38.12-38.7] 36 [35.55-38.5] <0.001 SAO2median [IQR] 94 [90-90] 95 [90.25-97] 93 [88.5-95.5] 0.119 pHmedian [IQR] 7.45 [7.38-7.38] 7.46 [7.4-7.5] 7.4 [7.24-7.4] <0.001 PO2median [IQR] 67 [56-56] 66.5 [56-78] 67 [56.5-79.5] 0.806 PCO2median [IQR] 36 [32-32] 37.5 [33-43.75] 34 [30-41] 0.245 Lactmedian [IQR] 1.5 [1-1] 1.3 [0.83-1.9] 2.5 [1.4-4.15] <0.001 Bicmedian [IQR] 23.5 [21-21] 24.25 [21.43-28] 21 [17.35-23.25] 0.001 FiO2 (%)median [IQR] 21 [21-21] 21 [21-23.25] 24 [21-45] <0.001 other Acute organ disfunctionyes 39 (43.3) 16 (64) 23 (35.4) 0.021 Apache score (%)median [IQR] 19 [12.5-12.5] 14.65 [12.12-20.38] 32 [20-39] <0.001 Days hospitalizedmedian [IQR] 5 [2-2] 6 [4-7] 2 [1-6] 0.003 treatment at baseline Diuresis (cc)median [IQR] 900 [600-600] 1000 [700-1200] 450 [200-1025] <0.001 Steroidsyes 16 (15.8) 4 (14.8) 12 (16.2) 1.000 Vasopressorsyes 18 (17.8) 13 (48.1) 5 (6.8) <0.001 Antibioticsyes 101 (100) 27 (100) 74 (100) 1.000 Fluid therapyyes 101 (100) 27 (100) 74 (100) 1.000

(46) 26.7% of all patients died during hospital stay and are counted as treatment non responder, 73.3% of all patients survived the sepsis and are counted as treatment responder.

(47) 53% off all patients presenting with sepsis had an non-normal PENK value >80 pmol/L (99 percentile), indicating PENK not to be a marker for the infection.

(48) Results of Clinical Study

(49) PENK highly correlated to creatinine clearance (r=0.74, p <0.0001, FIG. 4).

(50) PENK diagnoses kidney dysfunction:

(51) Kidney dysfunction was defined based on the RIFLE criteria (Venkatamaran and Kellum, 2007). Patients were counted as kidney dysfunction if any of the RIFLE classification factors was fulfilled. Within the study cohort, we determined the RIFLE within 90 subjects at day 1 (presentation at ED), 39 patients fulfilled RIFLE classification (had risk of kidney disease, kidney injury, kidney failure loss of kidney function or end-stage kidney disease) and 51 patients had no kidney dysfunction. Increased PENK was significantly (p=<0.0001) correlated with kidney dysfunction (AUC: 0.868). (FIGS. 5 and 6)

(52) To compare the diagnostic value for kidney dysfunction, we used NGAL as reference marker (Soni et al, 2010). NGAL was measured, using a commercially ELISA (NGAL Elisa kit, Bioporto, Gentofte, Denmark).

(53) NGAL, like PENK, was significantly increased in patients with kidney dysfunction (p<0.0001), the AUC for diagnosis of kidney dysfunction was 0.809. (FIGS. 7 and 8)

(54) Comparing PENK and NGAL showed a strong superiority of PENK vs NGAL for diagnosis of kidney dysfunction: the Chi2 value of PENK was 45.32 vs. 32.21 for NGAL, indicating a 40% improvement of diagnostic quality (specificity and sensitivity) by PENK. (Table 7)

(55) TABLE-US-00009 TABLE 7 Model Model N Events Chi2 d.f. LR p-value C index [95-CI] PCT 76 34 13.02 1 0.00031 0.721 [0.602,0.839] Apache 90 39 28.58 1 <0.00001 0.778 [0.681,0.874] NGal 90 39 32.21 1 <0.00001 0.809 [0.723,0.896] PENK 90 39 45.32 1 <0.00001 0.868 [0.796,0.94]
Initial PENK is highly prognostic.

(56) We correlated the initial PENK value with the in hospital mortality and compared PENK with APACHE 2 sepsis score (see Knaus et al, 1985, 2001) and creatinine clearance. PENK is highly prognostic for sepsis outcome (see FIG. 9) and comparable to APACHE 2 score (AUC/C index 0.744 (PENK) and 0.783 (Apache). There is a significant added information if PENK and APACHE 2 are combined (combined AUC: 0.794 FIG. 10). PENK is substantially stronger in prognosis than the creatinine clearance (AUC 0.638). Surprisingly, the prognostic value of PENK was stronger after the first day of ICU-treatment (AUC 0.79).

(57) Cut Off-Analysis for in hospital death prognosis using baseline sample and 1 sample after 1 day of ICU treatment.

(58) Since the prognostic power of PENK was further improved one day after starting ICU treatment, we analyzed the PENK in serial measurements of day before ICU-treatment and 1 day after starting ICU treatment. To illustrate the clinical performance, we used a simple cut off analysis at a cut off value of 100 pmol/L.

(59) If patients are below the cut off at hospital presentation and remain below the cut off after initiating ICU treatment, the mortality was 11% (well treated before and during hospitalization). If PENK was above the cut off at both time points, the mortality was about 5 times higher (52.5%) (not responding to treatment) and if patients present with PENK values above 100 pmol/1 and reducing their PENK levels below 100 pmol/1 during ICU treatment the mortality was 0 (treatment responder). These data indicate a strong association of PENK and treatment success, supporting its use for therapy follow up (serial testing).

(60) TABLE-US-00010 TABLE 8 mortality N patients died vs all PENK >100 pmol/l presentation and first 52.5% 21/40 day after ICU treatment PENK >100 pmol/l at presentation and 0% 0/7 <100 pmol/l first day after ICU treatment PENK <100 pmol/l at presentation and 11% 6/54 first day of ICU treatment

(61) FIG. 11a-d: examples of patient follow up measurements.

(62) a) A patient (survivor) with initial PENK <100 pmol/L and remained <100 pmol/L during hospital stay.

(63) b) A patient (died during hospital stay) with initial PENK >100 pmol/L and was not reduced to values <100 pmol/L.

(64) c) A patient (died during hospital stay) with initial PENK >100 pmol/L and was not reduced to values <100 pmol/L.

(65) d) A patient (survivor) with initial PENK>100 pmol/L, the PENK value declined to values <100 pmol/L within one day of ICU treatment.

Example 5

The use of serial measurement of PENK

(66) In the patient population described in example 4 (patients with sepsis, severe sepsis or septic shock) plasma PENK was measured on the day of admission and on the following day (day 1). Using a simple cut-off value of 100 pmol/L, which is close to the 99th percentile of the normal range, the population was segmented in two groups (above and below 100 pmol/L) and the corresponding 7 day survival rates were depicted in Kaplan-Meier-Plots (FIGS. 16 a) and b)). Patients with a PENK concentration below 100 pmol/L on the day of admission, whose PENK concentration remained below 100 pmol/L on day 1, had a high survival rate of 87%, whereas, when their PENK concentration increased over 100 pmol/L on day 1, the survival rate was lowered to 67%. In contrast, patients with a PENK concentration above 100 pmol/L on the day of admission, whose PENK concentration remained above 100 pmol/L on day 1, had a poor survival rate of only 50%, whereas, when their PENK concentration decreased below 100 pmol/L on day 1, the survival rate was 100%.

Example 6

(67) Using the plasma PENK concentrations determined in the patient population described in example 4 (patients with sepsis, severe sepsis or septic shock) on the day of admission, it was analyzed by multivariable linear regression analysis, which parameters/variables determine to which extent the PENK concentrations. In FIG. 17 the partial R2 are depicted. The analysis demonstrates that measures of kidney function (in the case shown creatinine clearance) are by far the strongest determinants for PENK concentrations.

(68) TABLE-US-00011 TABLE 9 Association of variables determined in the patient population as described in example 4 on the day of admission with the 7 day mortality. Variablemedian [IQR) deaths within 7 day all 7 days survivor (n = 101) (n = 28) (n = 73) p-value PENK (pmol/L) 87 [50-205) 209 [77-499) 75 [47-124) <0.001 Creatinine clearance 48 [23-77) 33 [15-69) 56 [29-81) 0.071 (ng/mL) Apache score (points) 16 [13-21) 23 [18-27) 14 [12-18) <0.001
PENK in males

(69) Using PENK as prognostic marker, PENK at first day (presentation at ED) was even stronger in prognosis of in hospital death in the male population (AUC 0.849, FIG. 12), a combination of PENK and Apache resulted in an AUC of 0.89 vs 0.837 Apache alone (FIG. 13). The combination of PENK and creatinine clearance generated a superior prognostic value of AUC 0.91 vs 0.721 for creatinine clearance alone (FIG. 14). As for the whole patient population, the prognostic value of PENK was stronger after the first day of ICU-treatment (day 2, AUC 0.872).

BRIEF DESCRIPTION OF FIGURES

(70) FIG. 1: shows a typical ProEnkephalin dose/signal curve. Standard curve proEnkephalin

(71) FIG. 2: frequence distribution of Pro Enkephalin in a healthy population (n=4211) The mean value of PENK was 44.7 pmol/L, standard deviation=1.27, the 99percentile (upper normal range) was 80 pmol PENK/L. FIG. 2 shows the LN values of PENK.

(72) FIG. 3: Correlation of creatinine clearance vs. PENK in healthy subjects. Y axis: quartiles of Creatinine Clearance, x axis: quartiles of PENK.

(73) FIG. 4: PENK highly correlated to creatinine clearance (r=0.74, p <0.0001).

(74) FIG. 5: Increased PENK was significantly correlated with kidney dysfunction.

(75) FIG. 6: A receiver/operator curve (ROC) for Pro-Enkephalin and the diagnosis of Kidney Dysfunction according the RIFLE criteria (see above). The area under the curve (AUC) was 0.868, indicating a strong diagnostic power of Pro-Enkephalin for Kidney Dysfunction.

(76) FIG. 7: Increased NGAL was significantly increased in patients with kidney dysfunction. The normal ranges of NGAL (range 0.037-0.106 g/mL; http://www.bioporto.com/products/bioporto_diagnostics/ngal_elisa_kits/ngl_rapid_elisa_kit_ce_ivd) is indicated by a shadowed area in the graph.

(77) FIG. 8: A receiver/operator curve (ROC) for NGAL and the diagnosis of Kidney Dysfunction according the RIFLE criteria (see above). We used NGAL as a reference marker for Kidney dysfunction. The AUC was 0.809, substantially lower than for Pro-Enkephalin (AUC 0.868, FIG. 6), indicating the incremental value of Pro-Enkephalin.

(78) FIG. 9: PENK is highly prognostic for sepsis outcome

(79) FIG. 10: There is a significant added information if PENK and APACHE 2 are combined

(80) FIG. 11a): A patient (survivor) with initial PENK <100 pmol/L and remained <100 pmol/L during hospital stay

(81) FIG. 11b): A patient (died during hospital stay) with initial PENK >100 pmol/L and was not reduced to values <100 pmol/L

(82) FIG. 11c): A patient (died during hospital stay) with initial PENK >100 pmol/L and was not reduced to values <100 pmol/L

(83) FIG. 11d): A patient (survivor) with initial PENK >100 pmol/L, the PENK value declined to values <100 pmol/L within one day of ICU treatment

(84) FIG. 12: PENK at first day (presentation at ED) was even stronger in prognosis of in hospital death in the male population

(85) FIG. 13: A combination of PENK and Apache resulted in an AUC of 0.89 vs. 0.837 Apache alone

(86) FIG. 14: The combination of PENK and creatinine clearance generated a superior prognostic value of AUC 0.91 vs. 0.721 for creatinine clearance alone

(87) FIG. 15: FIG. 15 A/B: Concentrations of plasma PENK (A) and NGAL (B), respectively, in septic patients categorized by grade of acute kidney dysfunction. 0=no kidney dysfunction; R=Risk; I=Injury; F=Failure; L=Loss. The categories are defined as follows (http://en.wikipedia.org/wiki/Acute_kidney_injury): Risk: GFR decrease >25%, serum creatinine increased 1.5 times or urine production of <0.5 ml/kg/hr for 6 hours; Injury: GFR decrease >50%, doubling of creatinine or urine production <0.5 ml/kg/hr for 12 hours; Failure: GFR decrease >75%, tripling of creatinine or creatinine >355 mol/l (with a rise of >44) (>4 mg/dl) OR urine output below 0.3 ml/kg/hr for 24 hours; Loss: persistent AKI or complete loss of kidney function for more than 4 weeks. Normal ranges of PENK (see FIG. 2) and NGAL (range 0.037-0.106 g/mL; http://www.bioporto.com/products/bioporto_diagnostics/ngal_elisa_kits/ngal_rapid_elisa_kit_ce_ivd) concentrations are indicated by shadowed areas in the graphs. The figure demonstrates that NGAL concentrations are massively elevated in septic patients even when they have no kidney dysfunction, whereas this is not the case for PENK.

(88) FIG. 16: Survival rates of critically ill patients depending on their plasma PENK concentrations on the day of admission and on the next day (day 1). Panel A: On the left hand side, the Kaplan-Meier-Plot is shown for those patient subpopulations with a PENK concentration on admission of above and below 100 pmol/L, respectively. On the right hand side the Kaplan-Meier-Plot is shown for those patient subpopulations with a PENK concentration on day 1 of above and below 100 pmol/L, respectively, who had a PENK concentration below 100 pmol/L on the day of admission. Panel B: On the left hand side, the

(89) Kaplan-Meier-Plot is shown for those patient subpopulations with a PENK concentration on admission of above and below 100 pmol/L, respectively. On the right hand side the Kaplan-Meier-Plot is shown for those patient subpopulations with a PENK concentration on day 1 of above and below 100 pmol/L, respectively, who had a PENK concentration above 100 pmol/L on the day of admission.

(90) FIG. 17: Multivariable linear regression predicting PENK. Note: BP, Creatinine and Urea were left out due to high correlation with MAP or creatinine clearance. The linear regression was calculated using the variables listed as follows: Log(PENK) =a*CreaClearance+b* Cardiovasc+c*MAP+etc . Partial R2 gives the degree up to which each variable contributes to PENK, i.e. Crea Clearance is strongest and has a partial R2 of slightly above 0.15, i.e. crea clearance accounts for about 15% of the variability that you observe in PENK. Importantly, age, gender, etc. do not have a significant influence on PENK concentrations.