DIAGNOSTIC METHOD FOR DISORDERS USING COPEPTIN

Abstract

Disclosed herein is the use of copeptin as a diagnostic marker for the determination of the release of vasopressin, especially in connection with disorders associated with non-physiological alterations of vasopressin release from the neurohypophysis, especially for detection and early detection, diagnosing and monitoring of the course of cardiovascular diseases, renal and pulmonary diseases as well as shock, including septic shock, sepsis and diseases/disorders of the central nervous system and neurodegenerative diseases.

Claims

1. A diagnostic in vitro method for the diagnosis and monitoring of disorders associated with or caused by non-physiological alterations of vasopressin release from the neurohypophysis by determining in a sample of a body fluid of the patient the amount of copeptin and/or a pathophysiologically occurring precursor, splice variant, fragment or posttranslationally modified form of copeptin displaying copeptin immunoreactivity, and associating the determined amount of copeptin or copeptin immunoreactivity with the presence and/or course and/or severity and/or prognosis of such disorder.

2. The method of claim 1, wherein said disorder associated with or caused by non-physiological alterations of vasopressin release from the neurohypophysis is selected from the group consisting of chronic or congestive heart failure, cardiac arrest, cardiac shock, cardiac infarction, acute myocardial infarction, arterial hypertension, cardiac surgery; cirrhosis; pulmonary disorders; kidney (renal) diseases as polycystic kidney disease; Diabetes insipidus; forms of hyponatremia, forms of syndrome of inappropriate antidiuretic hormone secretion; hemorrhage, edema-forming states, inflammatory diseases, trauma, burns, infectious complications thereof and sepsis, severe sepsis and septic shock; and diseases/disorders of the central nervous system (CNS).

3. The method of claim 1, wherein the copeptin immunoreactivity is an immunoreactivity which can be determined by using a sandwich immunoassay using a first specific binder recognizing an amino acid sequence present in SEQ ID NO:1, and a second specific binder recognizing an amino acid sequence present in SEQ ID NO:2.

4. The method of claim 3, wherein said specific binders are polyclonal and/or monoclonal antibodies.

5. The method of claim 3, wherein said immunoassay is a heterogenous immunoassay using a first immobilized specific binder and a second solubilized specific binder which carries a detectable label or which can be selectively labeled by reaction with a labeled marker molecule.

6. The method of claim 3, wherein said immunoassay is a homogeneous immunoassay.

7. The method of claims 1, wherein the biological fluid is selected from serum, plasma, blood or (cerebrospinal fluid; CSF).

8. The method of claim 2, wherein said disorder is selected from the group consisting of sepsis, cardiac infarction, chronic heart failure and increased arterial blood pressure.

9-12. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0044] FIG. 1 shows the standard curve for the sandwich-immunoassay for copeptin immunoreactivity using the peptide PAY33 as a standard material.

[0045] FIG. 2 shows the concentration of copeptin immunoreactivity in samples of healthy individuals and different groups of patients (sepsis, cardiac infarction and increased arterial blood pressure).

[0046] FIG. 3 shows the vasopressin prohormone amino acid sequence (one-letter code; cf. also SEQ ID NO:4).

[0047] FIG. 4 shows the concentrations of copeptin immunoreactivity in samples of patients with CHF (chronic heart failure) classified according to the NYHA classification system.

MATERIALS, METHODS AND MEASUREMENTS

EXAMPLE 1

[0048] Peptide Synthesis

[0049] Peptides were synthesized and their quality was controlled by mass spectrometry and reversed phase HPLC and lyophiliysed in aliquots (Jerini AG, Berlin, Germany) according to standard procedures known to the person skilled in the art. The amino acid sequences of the peptides are the following (numbers refer to corresponding positions in the human provasopressin-neurophysin 2-copeptin-precursors (positions 132-147 and 149-164):

TABLE-US-00003 PATV 17 (132-147 + N-terminal cystein residue): [Sequence ID 1] CATQLDGPAGALLLRLV, PLAY 17 (149-164 + N-terminal cystein residue): [Sequence ID 2] CLAGAPEPFEPAQPDAY, Standard peptide PAY 33 (132-164) [Sequence ID 3] ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY.

EXAMPLE 2

[0050] Conjugation and Immunization

[0051] Peptides of Sequence IDs 1-2 were conjugated to the carrier protein KLH (keyhole limpet hemocyanin) by MBS (-Maleimid-obenzoyl-N-hydroxysuccinimid ester) according to the protocols for “NHS-esters-maleimide crosslinkers” by PIERCE, Rockford, Ill., USA. Sheep were immunized receiving 100 μg of conjugate (μg according to the peptide content of the conjugate) and subsequently 50 μg of conjugate every four weeks (quantitiy related to the peptide content of the conjugate). Starting at month 4 after immunization every four weeks 700 ml of blood were withdrawn from every sheep and antiserum was gained by centrifugation. Conjugation, immunizations and production of antisera were done by MicroPharm, Carmerthenshire, UK.

EXAMPLE 3

[0052] Purification of Antibodies

[0053] The polyclonal antibodies from sheep were purified using ligand specific affinity purification. For that step the peptides PATV 17 and PLAY 17 were linked to SulfoLink-Gel supplied by Pierce (Boston, USA). The binding occurred according to the protocol of Pierce. 5 mg of peptide were added per 5 ml of gel.

[0054] In summary, columns were washed three times with 10 ml elution buffer (50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate, 0.1% Tween, pH 6.8). 100 ml of sheep antiserum were filtered using a filter diameter of 0.2 μm and added to the column material, which had been transferred from the column to a beaker with 10 ml binding buffer. The material was incubated over night at room temperature by gentle rotation. The material was transferred to empty columns (NAP 25, Pharmacia, emptied). The eluates were discarded. Subsequently the columns were washed with 250 ml protein-free binding buffer (protein content of washed eluate <0.02 A 280 nm): Elution buffer was added to the washed columns and fractions of 1 ml were collected. The protein content of each fraction was determined by the BCA-method (according to the protocol of PIERCE, Rockford, Ill., USA). Fractions of a protein content >0.8 mg/ml were pooled. After determination of protein content 39 mg of anti-PATV 17 antibody 0413-pAk and 103 mg of anti-PLAY 17 antibody 0417-pAk were gained.

EXAMPLE 4

[0055] Tagging

[0056] The anti-PLAY 17 antibody 0417-pAk was treated as follows: 500 μl of affinity purified antibodies generated were rebuffered in 1 ml 100 mM potassium phosphate buffer (pH 8.0) via a NAP-5 gel filtration column (Pharmacia) according to the protocol of Pharmacia. The protein concentration of antibody solution was 1,5 mg/ml.

[0057] For the tagging with a chemiluminescent marker 10 μl of MA70-Akridinium-NHS-ester (lmg/ml; Hoechst Behring) were added to 67 μl of antibody solution and incubated for 15 minutes at room temperature. Then 423 μl of 1 M glycin was added and incubated for 10 minutes. The solution was rebuffered in 1 ml solvent A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4) using a NAP-5 gel filtration column according to the protocols of Pharmacia. For final elimination of unbound label a gel filtration HPLC was done (Column: Waters Protein Pak SW300). The sample was added and chromatographed at a flow rate of 1 ml/minute in solvent A. The flow was continuously monitored in a UV-meter at wave length of 280 and 368 nm to determine the degree of tagging. The absorption ratio 368/280 nm of labelled antibody was 0,1. The fractions containing monomeric antibodies were collected (retention time 8-10 minutes) and taken up in 3 ml 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0,1% sodium azide, pH 7.4)

EXAMPLE 5

[0058] Coupling

[0059] The anti-PATV 17 antibody 0413-pAk was immobilized on irradiated 5 ml polystyrol tubes (Greiner, Germany). For that procedure the antibody solution was diluted to a protein concentration of 6.6 μg/ml with 50 mM Tris, 100 mM NaCl, pH 7.8. 300 μl of diluted protein solution per tube were pipetted. These were incubated for 20 hours at 22° C., the solution was removed. Then 4.2 ml of a 10 mM sodium phosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5 solution were added to each tube. After 20 hours the solution was removed and the tubes were dried in a vacuum drier.

EXAMPLE 6

[0060] Immunoassay

[0061] The following assay buffer was used: 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% unspecified sheep IgG, 0.1% sodium azide, pH 7.4.

[0062] The copeptin concentration of EDTA-plasma of healthy individuals and patients of various diseases/diseases mentioned above was determined, heart diseases and diseases of the circulation in particular.

[0063] As a standard material chemically synthesized peptide (peptide PAY 33) was used which corresponds to positions 132-164 of vasopressin-neurophysin 2-copeptin precursor. The standard was diluted in normal horse serum (Sigma).

[0064] In the test tubes 100 μl of standards or sample as well as 100 μl of assay buffer was pipetted. The tubes were incubated for two hours at 22° C. using gentle rotation. After washing 4 times with 1 ml of washing buffer (0.1% Tween 20), the supernatant was discarded. Then 200 μl of assay buffer, containing 1 million RLU (relative light units) of MA70-tagged antibody was added and incubated for a further two hours under gentle rotation at 22° C. After washing 4 times with 1 ml of washing buffer (0.1% Tween 20), the chemiluminescence bound to the tube was determined in a luminometer (Berthold, LB952T, basic reagents Brahms AG). Using the software MultiCalc (Spline Fit) the concentrations of the samples were determined.

EXAMPLE 7

[0065] Determination of Copeptin Concentration

[0066] The term copeptin immunoreactivity describes the amount of substrate detected by the developed sandwich immunoassay. The sandwich immunoassay uses antibodies raised against positions 132-147 and 149-164 of the vasopressin-neurophysin 2-copeptin-precursor for detection of the substrate. A typical standard curve for the developed assay is described in FIG. 1. Using the assay concentrations above 50 pg/ml copeptin immunoreactivity in plasma or serum can be determined quantitatively.

EXAMPLE 8

[0067] Concentration of Copeptin Immunoreactivity in Healthy Individuals and State of Disease

[0068] Serum and plasma of healthy individuals and patients suffering from various diseases comprising sepsis, cardiac infarction and increased arterial blood pressure were analysed (FIG. 2): The copeptin immunoreactivity was determined. Compared to healthy individuals copeptin immunoreactivity was surprisingly increased in state of disease. Healthy individuals showed a median of about 13 pg/ml, sepsis patients a median of about 150 pg/ml, in cardiac infarction the median was about 129 pg/ml and in increased arterial blood pressure the median was about 459 pg/ml.

EXAMPLE 9

[0069] Concentration of Copeptin Immunoreactivity in Patients with Chronic Heart Failure (CHF) of NYHA Classes I to IV

[0070] In serum and plasma samples of a total of 348 CHF patients (25 in NYHA class I; 124 in NYHA class II; 127 in NYHA class III; 72 in NYHA class IV; see Table 1) copeptin levels were determined using the assay described above. The results are shown in diagrammatic form in FIG. 4. As can be seen, the medians of the copeptin concentration in pg/ml for the different classes showed a clear tendency to increase in that the median for patients of NYHA class I was found to be 20.30 pg/ml, class II 33.25 pg/ml, class III 49,60 pg/ml, and class IV 85.80 pg/ml (see the statistical data in Table 1 below).

TABLE-US-00004 TABLE 1 NYHA I NYHA II NYHA III NYHA IV Number of 25 124 127 72 values Median 20.30 33.25 49.60 85.80 Mean 27.00 45.32 63.91 184.7 Lower 95% CI 17.45 38.79 55.29 118.9 of mean Upper 95% CI 36.56 51.86 72.54 250.5 of mean CI = confidence interval

[0071] The finding that there is a close correlation of the severity of CHF with the copeptin levels in plasma makes copeptin a biomarker candidate for use in the diagnosis (positive or negative diagnosis) of CHF, the monitoring of the course and evolution of CHF and the monitoring and control of a CHF therapy. Further, in view of current attempts to evaluate the usefulness of vasoporessin receptor antagonists in the therapy of heart failure [27], the determination of copeptin in serum or. plasma samples of heart failure patients can allow the identification of such patients who would benefit more than others from a treatment with vasopressin receptor antagonists.

EXAMPLE 10

[0072] Stability of Copeptin Immunoreactivity

[0073] Copeptin immunoreactivity was found to be surprisingly stable in plasma and serum (Table 2). Table 2 shows the ex vivo stability of endogenous immunoreactive copeptin in serum and plasma of sepsis patients.

TABLE-US-00005 TABLE 2 Storage Recovery Sample (days/temperature) (%) Serum 1 d/4° C. 98.0% (n = 3) 2 d/4° C. 99.2% 1 d/RT 94.1% 2 d/RT 103.7% Plasma 1 d/4° C. 103.4% (n = 5) 2 d/4° C. 101.6% 1 d/RT 99.9% 2 d/RT 104.9%

[0074] Even after two days storage at room temperature (RT) no decrease in immunoreactivity could be detected.

[0075] Thus the ex vivo stability of copeptin immunoreactivity is surprisingly remarkably increased as compared to vasopressin.

LITERATURE

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