HK1099807B - Method for detecting the formation of endothelins for medical diagnosis, and antibodies and kits for carrying out one such method - Google Patents

Description

method for determining endothelin formation for medical diagnostic purposes, and antibodies and kits for carrying out said method

The invention relates to the determination of endothelin formation in the case of severe diseases for the purpose of medical diagnosis by determining peptide fragments of the corresponding pro-endothelin in the circulation (whole blood, plasma or serum), in particular the relatively long-lived C-terminal part peptide of pro-endothelin-1, more precisely in sepsis diagnosis, heart disease diagnosis, for example also in cancer diagnosis, more precisely generally in the diagnosis of such pathologies in which endothelin plays an important role in the course of the disease.

When simply used in this application, "endothelin" is the term first referring to endothelin-1 (ET-1). However, the corresponding expressions are often also common for other isoforms of endothelin, since the restriction to endothelin-1 does not seem to be generally necessary, and in a broad sense the invention should also relate to other endothelins.

In the present description, the term "diagnosis" is used in principle as a simplified generic term, which in particular shall also include prognosis/early prognosis and the concomitant process control of the treatment.

In particular, the assay is carried out by means of specific immunodiagnostic methods, in particular by means of a class of immunoassays (sandwich assays; competition assays, for example according to the SPALT or SPART principle) in which at least one labeled antibody is used.

Endothelin-1 (ET-1) is a21 amino acid peptide, which is the most potent vasoconstrictor known. Since its inception in 1988 by Yanagisawa and colleagues [ 27; the numbers in brackets relate to the attached list of references ] since their discovery, their biosynthesis, mode of action and association with disease have been studied extensively and are summarized in topical review articles [1, 7, 12, 17, 24 ]. There are three isoforms of endothelin (endothelin-1, endothelin-2, endothelin-3) encoded by different genes, with endothelin-1 being the most concentrated and most potent. Endothelin-1 is synthesized in endothelial cells, lung, heart, kidney and brain. The initial translation product of the human endothelin-1 gene is a peptide containing 212 amino acids, namely proendothelin-1 (SEQ ID NO: 1). During secretion, the short N-terminal signal sequence (amino acids 1 to 17) of proendothelin is cleaved off by a signal peptidase. The proendothelin obtained here is subsequently processed by a protease acting on the dibasic amino acid pair, furin, into a biologically inactive 38 amino acid-containing peptide, big-Endothelin (SEQ ID NO: 3), whereby finally mature, biologically active Endothelin-1 (SEQ ID NO: 2) is formed with the aid of Endothelin-converting enzyme (ECE). Endothelin acts by binding to specific receptors located on myocytes, cardiomyocytes and fibroblasts. This binding results in calcium efflux, phospholipase C activation and Na/K ATPase inhibition. In addition to vasoconstrictive effects, endothelin also has growth regulating properties.

In view of the large number and presumed physiological roles of endothelin, particularly endothelin-1, since its identification, a number of different assays have been developed for its immunodiagnostic determination and used to measure endothelin, particularly in human plasma. Such assays are the subject of many publications.

Increased plasma concentrations of endothelin-1 and macroendothelin have been described for a variety of disease conditions [17 ]. The diseases which belong to this group are cardiovascular diseases [1] (in particular pulmonary hypertension [21], atherosclerosis [13], congestive heart failure [25], myocardial infarction [20]), sepsis and septic shock [11, 22, 23], cancer [2, 3, 15, 18], etc.

Immunoassays for measuring endothelin in plasma samples (for review see [17 ]) are particularly of the radioimmunoassay type (using labeled endothelin-1 as competitor) or of the EIA/ELISA type, and these methods are intended only for the determination of endothelin or for the determination of endothelin-immunoreactivity. Here, RIA-type assays have low specificity and also include related peptides containing endothelin sequences.

However, endothelin-1 (ET-1) was found to have a very short residence time in the circulation, after 1-2 minutes it was already cleared from the circulation [6 ]. Since endothelin-1 is considered to be stable in blood and plasma [6], its distribution into other tissues and its rapid and high affinity binding to receptors are considered to be the most important reasons for short residence time. Thus, in certain tissues and body fluids, significantly higher concentrations of endothelin-1 can be measured than, for example, in plasma [1, 7 ]. In view of these circumstances, the correctness of the determination of ET-1 in plasma samples is seriously questioned [17 ]. That is, it is believed that the transient (perhaps only transient) ET-1 concentration that can be measured in plasma samples is not important for the physiological effects of endothelin (ET-1), whereas the sum of all free and bound (e.g., tissue and receptor bound) physiological ET-1 concentrations present in an organism has a much greater correlation.

The determination of the precursor of ET-1, the so-called macroendothelin ("bigET-1"; SEQ ID NO: 3), has the advantage over the determination of ET-1 that the residence time of the "macroendothelin" in the circulation is significantly longer than the residence time of the ET-1 released therefrom. Thus, in a series of studies, the true endothelin was not determined, but rather the "large endothelin" was determined. In particular, sandwich-type assays are used for their specific assay, which enable reliable differentiation of large endothelin-1 from processed ET-1 and other endothelins [4, 8, 10 ]. It was shown that in the case of certain diseases, the measured increase in ET-immunoreactivity could be attributed to large endothelin.

Selective measurement of large endothelin-1 is only a gradual improvement of the problem and does not actually solve the problem, since large endothelin can also be rapidly processed to endothelin in the blood circulation [1, 5, 9 ]. Thus, it also has a relatively short biological half-life (20-25 minutes) [10], and thus, large endothelin measurements measurable in plasma likewise represent only transient plasma concentrations, and do not reflect true physiologically acting endothelin concentrations. When measuring large endothelin-1, ET-1 that is physiologically formed under disease conditions but has been processed and bound in tissues or receptors is not included. Therefore, in the case of measuring large endothelin, the total amount of physiologically active endothelin is also underestimated. An attempt was made to specifically measure complementarily the C-terminal peptide fragment of large endothelin-1 (having amino acids 74 to 90 of proendothelin or amino acids 22 to 38 of large endothelin) formed in addition to ET-1 upon enzymatic cleavage of large endothelin-1, and the results showed that such a peptide is inferior in stability to ET-1 and thus is not suitable for measurement [10 ].

For the measurement of a range of proendothelin other than large endothelin, only one commercial competitive assay is known from the prior art (N-terminal region 18-50, commercially available from PhoenixPharmaceutics; the use for the diagnosis of sepsis is described in WO 00/22439). No information is disclosed on the stability and properties of the analyte to be measured with this assay.

It is an object of the present invention to develop an assay which is able to reflect the endogenous formation of macrophthaleine or endothelin, i.e. the total physiological concentration, and thus the action of endothelin more reliably than hitherto for the determination of ET or macrophthaleine in plasma.

One such method should be efficient and amenable to programming and be capable of providing reliable values for the physiological production of ET (ET-1) and/or its precursors in the context of a variety of different disease conditions, particularly in the context of sepsis or other disease conditions in which elevated values of endothelin play a role.

The object of the invention is achieved by determining for diagnostic purposes not ET or macroendothelin in whole blood, plasma or serum samples of human patients but a relatively long-lived pre-pro-endothelin-or pro-endothelin-partial peptide, in particular the C-terminal part peptide, which does not contain the ET or macroendothelin sequence and which contains at least amino acids 168-212 of pre-pro-endothelin-1.

Claim 1 relates to the principle of the invention. Advantageous and presently preferred embodiments of the invention are described in the dependent claims.

The present invention is based on the applicant's experimental studies in which it can be shown that such a fraction of pro-endothelin is not a direct precursor of endothelin, comprising long-lived peptides suitable for measurement purposes, which can be measured reliably and with high clinical value in blood samples.

Physiologically, endothelin-1 is formed by processing of a larger precursor molecule, namely proendothelin (SEQ ID NO: 1) or secreted proendothelin obtained therefrom. During such processing, in addition to the large endothelin (and endothelins derived therefrom), other peptides must be formed in initial stoichiometric amounts, which have never been the subject of scientific research to date and are unknown with respect to their possible other processing and stability. At the outset of applicants' study, it is desirable to be able to demonstrate that at least one putative additional peptide-cleavage product is present in a blood sample (whole blood, plasma or serum sample) and has proven to be relatively stable and thus may be suitable as a measure of physiological formation of endothelin, regardless of the actual endothelin concentration measurable in plasma.

Thus, measuring such lysate may be the sought method for determining the physiological concentration or production of endothelin, which is referred to in the claims as determining "endothelin formation". The term relates to the physiological concentration of endothelin-1 formed in connection with a disease corresponding only to the amount of previously processed pro-endothelin or pro-endothelin, assuming only one, i.e. a single known, formation pathway for the formation of endothelin-1 from pro-endothelin. If a partial peptide formed at the same stoichiometric concentration, except for large endothelin or endothelin, is a stable "metabolic waste" that neither binds to the receptor nor partitions into the tissue, it must be retrieved in the circulation. It is not necessary to involve a certain physiological mechanism for this reason, and it is also possible to consider "determining/measuring the formation of endothelin" as measuring "secretory activity" or "production of secretory pro-endothelin".

In the present application, the peptide fragments to be assayed are characterized by a "longevity". The term is intended to mean that the peptide fragments to be determined have a significantly longer residence time in the circulation (in whole blood) than the endothelin or macroendothelin fragments. In particular, the term "long-lived" is used to indicate that such peptide fragments do not undergo further rapid proteolytic cleavage in whole blood and plasma obtained therefrom, and are cleared from circulation or metabolism at a significantly slower rate than the rate of binding of endothelin to the receptor and the rate of proteolytic cleavage of the cleavable segment.

Due to said longer stability or "longevity", in the presence of such fragments, information about the secretory activity that has elapsed is stored for a period of time that is at least suitable for problem-free measurement. If, for example, the endothelin precursor is supposed to be released in a single, short decantation, the amount of "long-lived" fragments measurable after a certain time corresponds to the amount initially decanted, only subtracted by the amount associated with the physiological half-life of the peptide fragments to be measured in the circulation. On the other hand, if it is assumed that, for example, endothelin precursors are produced more or less continuously during the onset of the disease, the physiological production of past precursors is reflected cumulatively in the measurable concentration of peptide fragments long-lived in the above sense, but the decrease in the concentration of peptide fragments occurring over the same time in accordance with their physiological clearance rate is subtracted again. Active endothelin or its precursors, i.e. macroendothelin, are already processed or cleared from the circulation in the same time and for example bind to receptors and are therefore no longer measurable. The longer the peptide fragment is, or the smaller its clearance rate, the less the influence of the measurement time point on the accuracy of the determination of the "formation" of the above-mentioned biomarker (i.e. endothelin). In this case, a constant concentration over a longer period of time indicates that an equilibrium is reached between formation and scavenging. If the concentration is reduced, this may indicate that secretion of the precursor molecule (e.g. proendothelin) has ceased, e.g. because the molecular reservoir is depleted, and the change in concentration to be observed is solely determined by the rate of clearance.

Thus, measurement of long-lived peptide fragments without known physiological function provides results that are not only quantitatively but also qualitatively different from the measurement of a relatively short-lived active peptide or its precursor that is also relatively short-lived.

Studies by the applicant, which will be described in detail below, have shown that the above-described method provides fruitful results in the context of determining endothelin formation.

The studies carried out and the most important results of these studies will be explained more precisely in the following, in which reference will be made to the appended figures.

Description of the drawings:

FIG. 1: typical standard curves for a currently preferred sandwich assay described more accurately in the experimental section for the determination of C-terminal proendothelin peptide sequences in human plasma, using two antibodies which bind the amino acid sequences corresponding to positions 168-181 and 200-212 of proendothelin-1;

FIG. 2: figure, which shows that no significant loss of immunoreactivity occurred in the assay according to figure 1 in case of EDTA-plasma samples from sepsis and heart disease patients left at room temperature for 12 hours;

FIG. 3 a: plasma measurements of 5 groups of human patients with different diseases/diagnoses compared to measurements of apparently healthy individuals; dotted lines represent the maximum found in healthy individuals (100% specific line based on healthy controls);

FIG. 3 b: a graph corresponding to figure 3a for another 4 groups of patient sera.

The method of the invention, in its most general aspect, involves the determination of relatively long-lived peptide fragments of pro-endothelin-1, which do not contain the amino acid sequence of endothelin-1 or its precursors, i.e., macroendothelin, in a patient's whole blood, plasma or serum sample (i.e., circulation) when severe disease conditions exist, in order to indirectly determine the formation of endothelin, particularly endothelin-1. According to a preferred embodiment, the peptide fragment to be determined is a C-terminal fragment to which two antibodies bind which bind to peptides having amino acid sequences corresponding to positions 168-181 and 200-212 of proendothelin-1.

For the practical transformation of the present invention, it is particularly preferred to plan for a non-competitive sandwich assay, for example of the type used for continued in-depth studies and described more precisely below.

Compared to competitive immunoassays, noncompetitive sandwich immunoassays (double-sided immunoassays) have many advantages, including being able to be better designed than solid-phase assays (heterogeneous assays), being able to be more robust in terms of operability, being able to provide measurement results with higher sensitivity, and also being better suited for automation and batch measurements. In addition, the non-competitive sandwich immunoassay is able to provide additional information compared to competitive immunoassays that work with only one type of antibody, which is accomplished by the sandwich immunoassay recognizing only the molecules or peptides in which there are two binding sites on the same molecule for the antibody used to form the sandwich.

In principle, the antibodies that can be used can be any suitable monoclonal and/or polyclonal antibody, but among these, affinity-purified polyclonal antibodies are presently preferred.

Particularly preferred are antibodies obtained by immunizing an animal, particularly sheep, with an antigen comprising a synthetic peptide sequence corresponding to the short amino acid sequence of proendothelin-1 and having an additional cysteine residue at the N-terminus. In the experimental section that follows, antibodies that bind to the amino acid sequences at positions 161-181 and 200-212 or their use in assays are described in particular. However, it is also possible to use further antibodies within the scope of the investigation, which bind to the positions 184-203 and 136-148, respectively. The additional results obtained using these other antibodies in the measurements are only discussed in general terms in this application.

In a preferred embodiment, the method is performed as a heterogeneous (heterogener) sandwich immunoassay, wherein one antibody is immobilized on an arbitrary solid phase, such as the wall of a Coated test tube (e.g.made of polystyrene; "Coated Tubes"; CT), or on a microtiter plate, e.g.made of polystyrene, or on a particle, e.g.a magnetic particle, and the other antibody has a residue which is a directly detectable label or enables selective attachment to a label, and facilitates detection of the resulting sandwich structure. A time-delayed or subsequent immobilization using a suitable solid phase is also possible.

In principle, all labeling techniques which can be used in the described types of assays, the useful radioisotopes, enzymes, fluorescent, chemiluminescent or bioluminescent markers belonging thereto, and direct labeling by visually detectable color, such as gold atoms and dye particles, can be used, as they are used in particular for the so-called Point-of-Care (POC) test or rapid tests for assays in whole blood samples. In the case of heterogeneous sandwich immunoassays, both antibodies may also have parts of a detection system of the type described below in relation to the monophasic assay (homogenen assays).

It is therefore also within the scope of the invention to arrange the method of the invention for rapid testing.

Furthermore, the method of the present invention may be arranged as a single phase method, wherein a sandwich complex formed by the two antibodies and the peptide fragments to be detected is kept suspended in a liquid phase. In such a case, it is preferred to label both antibodies with parts of the detection system which enable the generation of a signal or trigger signal if both antibodies are integrated into a single sandwich. Such techniques may be arranged in particular as fluorescence enhancement or fluorescence quenching detection methods. A particularly preferred method of this type involves the use of detection reagents used in pairs, as described, for example, in US-A-4822733, EP-B1-180492 or EP-B1-539477 and the prior art cited therein. It enables measurements to be made directly in the reaction mixture that selectively include only the reaction product containing the two marker components in a single immune complex. See, by way of example, the trademarks(Time Resolved Amplified Cryptate Emission) orTechniques are provided which embody the teachings of the above-identified application.

In the applicant's studies it was demonstrated that the determination of the C-terminal peptide fragment of proendothelin-1 according to the present invention provides very meaningful and relevant measurements. As will be demonstrated hereinafter, this information is applicable not only to the diagnosis of sepsis but also to the diagnosis of heart disease and the diagnosis of cancer.

Furthermore, it is assumed that the assay method according to the invention can also be carried out particularly advantageously in the context of so-called multiparameter diagnostics and can be used not only in the field of heart disease diagnostics but also in the field of sepsis and cancer diagnostics. Further parameters determined here are, for example, cardiac parameters ANP, BNP, proANP, proADM or proBNP, or sepsis parameters, which are, for example, selected from the group consisting of anti-ganglioside antibodies, the protein procalcitonin, CA125, CA19-9, S100B, S100A-protein, LASP-1, soluble cytokeratin fragments, in particular CYFRA21, TPS and/or soluble cytokeratin-1 fragment (sCY1F), peptides Inflammin and CHP, further peptide prohormones, glycine-N-acyltransferase (GNAT), carbamoylphosphate synthase 1 (1) and C-reactive protein (CRP) or fragments thereof. In the case of the multi-parameter determination, the measurement results of a plurality of parameters are determined at the same time or in parallel and evaluated, for example, by means of a computer program which also makes use of diagnostically significant parameter correlations.

Hereinafter, the present invention will be explained in more detail by the description of the following: preparation of preferred assay components, implementation of preferred embodiments of sandwich-type assays, and results of assays for C-terminal peptide fragments in EDTA-plasma of control individuals as well as sepsis, heart disease and cancer patients obtained by using such assays.

Experimental part

A. Materials and methods

1. Synthesis of peptides

Three regions were selected (positions 168-181, 184-203, 200-212) from the known amino acid sequence of human proendothelin-1 (SEQ ID NO: 1). In each case, these regions were chemically synthesized as soluble peptides according to standard methods in a manner complementary to the N-terminal cysteine residue, purified, quality controlled by means of mass spectrometry and reverse phase HPLC, and lyophilized into aliquots (JERINI AG, Berlin, Germany). The amino acid sequences of these peptides are as follows:

peptide PCT15 (168-181+ N-terminal cysteine)

CRSSEEHLRQTRSET (SEQ ID NO：4)

The peptide PCW14 (200-

CSRERYVTHNRAHW (SEQ ID NO：5)

The peptide PNR20 (184-

NSVKSSFHDPKLKGKPSRER (SEQ ID NO：6)。

In addition, as a standard for calibrating the assay, the following peptides were synthesized:

standard peptide PSW44(169-212)

SSEEHLRQTRSETMRNSVKSSFHDPKLKGKPSRERYVTHNRAHW

(SEQ ID NO：7)。

2. Conjugation and immunization

The peptides PCT15 and PCW14 were conjugated to the carrier protein KLH (keyhole limpet hemocyanin) by means of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) (see the instructions "NHS-Esters-Maleimide Crosslevers", PIERCE, Rockford, IL, USA). Sheep were immunized with these conjugates according to the following protocol: initially, 100. mu.g of conjugate (based on the mass value of the peptide portion of the conjugate) was obtained for each sheep, and subsequently 50. mu.g of conjugate (based on the mass value of the peptide portion of the conjugate) was administered to each sheep every 4 weeks. From the 4 th month after the start of immunization, 700ml of blood was drawn every 4 weeks for each sheep, and antiserum was obtained therefrom by centrifugation. Conjugation, immunization and antisera were obtained by the company MicroPharm (Carmarthenshire, UK).

3. Purification of antibodies

In the 1-step method, peptide-specific antibodies are prepared from antisera obtained starting at month 4 after immunization.

For this purpose, the peptides PCT15 and PCW14 were first coupled on a SulfoLink gel (see the instructions "SulfoLink Kit", PIERCE company, Rockford, IL, USA). Here, 5mg of peptide per 5ml of gel are provided each time for coupling.

Affinity purification of peptide-specific antibodies from sheep antisera against these two peptides was performed as follows:

first, the peptide column was washed three times with 10ml each time of elution buffer (50mM citric acid, pH2.2) and binding buffer (100mM sodium phosphate, 0.1% Tween, pH6.8) alternately. 100ml of antiserum were filtered through 0.2 μm and incorporated into the column material present. For this purpose, the gel was quantitatively rinsed out of the column with 10ml of binding buffer. Incubate overnight at room temperature with shaking. The incubated mixture was quantitatively transferred to an empty column (NAP25, Pharmacia, empty). The effluent is discarded. Subsequently, the column was washed protein-free with 250ml of binding buffer (protein content of the wash eluate <0.02, a280 nm). Elution buffer was added to the washed column and fractions were collected in a volume of 1 ml. The protein content in each fraction was determined by means of the BCA-method (see the operating manual, PIERCE, Rockford, IL., USA). Fractions with protein concentration >0.8mg/ml were pooled. After protein determination of the pool by means of the BCA-method, 97mg of anti-PCT 15 antibody 0407-pAk and 60mg of anti-PCW 14 antibody 0410-pAk were harvested.

4. Marking

anti-PCW 14 antibodies 0410-pAk were treated as follows:

according to the instructions, 500. mu.l of the purified antibody was rebuffered into 1ml of 100mM potassium phosphate buffer (pH8.0) through NAP-5 gel filtration column (Pharmacia). The protein concentration of the antibody solution was measured to be 1.5 mg/ml.

For the chemiluminescent labeling of the antibodies, 67. mu.l of the antibody solution was spiked with 10. mu.l of MA 70-acridinium-NHS-ester (1 mg/ml; HOECHST Behring Co.) and incubated at room temperature for 15 minutes. Then, 423. mu.l of 1M glycine was added and incubated for another 10 minutes. Subsequently, the labeled impurities were rebuffered into 1ml of mobile phase A (50mM potassium phosphate, 100mM NaCl, pH7.4) by NAP-5 gel filtration column (Pharmacia) according to the instructions, and the low molecular weight components were removed therefrom. To separate the final residue of the label not bound to the antibody, gel filtration-HPLC (column: Waters Protein Pak SW300) was performed. The sample was applied and chromatography was performed with mobile phase a at a flow rate of 1 ml/min. The wavelengths 280nm and 368nm were measured with a flow photometer (Duchflu. beta. photometer). The absorbance at 368nm/280nm, which is a measure of the degree of antibody labeling, peaked at 0.10. Fractions containing monomeric antibody were collected (retention time 8-10 min) and collected in 3ml100mM sodium phosphate, 150mM NaCl, 5% bovine serum albumin, 0.1% sodium azide, pH 7.4.

5. Coupling of

anti-PCT 15 antibody 0407-pAk was treated as follows:

irradiated 5ml polystyrene vials (Greiner) were coated with purified antibody as follows: the antibody was diluted to a concentration of 6.6. mu.g/ml in 50mM Tris, 100mM NaCl, pH 7.8. In each vial, 300. mu.l of such solution was pipetted. The vials were incubated at 22 ℃ for 20 hours. The solution was aspirated. Each vial was then filled with 4.2ml of 10mM sodium phosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5. After 20 hours, the solution was aspirated. Finally, the vials were dried in a vacuum desiccator.

B. Implementation and evaluation of immunoassays

Assay buffers were prepared having the following composition: 100mM sodium phosphate, 150mM NaCl, 5% Bovine Serum Albumin (BSA), 0.1% non-specific sheep IgG, 0.1% sodium azide, pH 7.4.

The chemically synthesized peptide described above (peptide PSW44) was used as a standard material, which corresponds to position 169-212 of proendothelin-1. The peptide was serially diluted in horse normal serum (SIGMA corporation). The concentration obtained from weighing the peptide was attributed to the standard thus prepared.

The samples measured were EDTA-plasma from individuals who looked healthy, from patients with sepsis and from patients with different cardiovascular diseases.

In the test tube, 50. mu.l of standard or sample and 200. mu.l of assay buffer were transferred. Incubate at 22 ℃ for 2 hours with shaking. Then, wash 4 times with 1ml of wash solution (0.1% Tween20) per vial and let dry. Then, 200. mu.l of assay buffer containing 1 million RLU (relative light units) of MA 70-labeled antibody was transferred. Incubate at 22 ℃ for 2 hours with shaking. Then, the vials were washed 4 times with 1ml of washing solution (0.1% Tween20) per vial, drained and the chemiluminescence bound to the vials was measured in a luminometer (BERTHOLD, LB 952T; Basisreagen BRAHMS AG).

The concentration of the sample was read on a standard curve using the software multicalc (spline fit).

C. Results

The analytes measurable with the developed sandwich immunoassay (antibodies against positions 168-. A typical standard curve for the test developed is shown in figure 1. Using this test, it is also possible to determine Ct-proendothelin concentrations well below 50 pg/ml.

In order to examine whether the question of problems due to insufficient stability in the sample or the measurement solution must be taken into account when measuring the C-terminal peptide fragment, 5 sepsis plasmas were measured at room temperature each time at a fresh time and after 12 hours of storage. The results are summarized in fig. 2. It showed that after 12 days of storage, there was still approximately 93% of the immunoreactivity originally measured, with little change. This stability of the test is a great advantage from an operational point of view for diagnostics.

Using this test, plasma was measured in patients with heart disease as well as sepsis. The results obtained are shown in figures 3a and 3 b. For all the cardiac disease conditions studied, elevated values were found for normal controls. Likewise, elevated values are also found for patients with SIRS (systemic inflammatory response syndrome) and sepsis conditions. Here, the diagnostic sensitivity (when given 100% specificity based on healthy controls) increases with the severity of the disease: sepsis 32.3%, severe sepsis 65.5%, and septic shock 75%.

Essentially the same results were obtained as expected when the samples were measured using a modified assay in which one of the antibodies of the sandwich assay described above was replaced with an antibody recognizing amino acid 184-.

In contrast, if one of the antibodies used recognizes an amino acid sequence located closer to the N-terminal direction of proendothelin (32-52 or 136-148), an elevated measurement cannot be obtained relative to a healthy individual. This indicates that endothelin is not present in the measured plasma sample per se and is not only proteolytically processed to form large endothelin, but that the C-terminal sequence 93-212 released there is further cleaved, wherein at least one such cleavage site must lie within the range of amino acids 149-167. This information applies to the plasma of patients with the disease under study. However, it cannot be excluded that e.g.the complete C-terminal fragment 93-212 is still obtained in other patient groups and that a measurement of its selectivity can provide diagnostically relevant results.

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Claims (14)

1. Kit for the in vitro determination of the formation of endothelin in severe diseases for immunodiagnostics, determining the amino acid sequence of SEQ ID NO: 1 comprising a peptide sequence within amino acid range 168-212 of pre-proendothelin-1, characterized in that the kit comprises, in addition to conventional reagents, buffers and washing solutions as standards, the amino acid sequence SEQ ID NO: 1, comprising the amino acid sequence of amino acid SEQ ID NO: 7, said serious disease being a systemic inflammatory response syndrome, a sepsis condition, a cardiopathy condition and cancer.

2. The kit according to claim 1, characterized in that at least one antibody is a labeled antibody.

3. Kit according to claim 1 or 2, characterized in that the determination is performed by a competitive immunoassay or a sandwich immunoassay.

4. Kit according to claim 1 or 2, characterized in that it comprises an antibody pair which binds to a polypeptide present in the C-terminal fragment SEQ ID NO: 7 selected from the group consisting of peptide sequences having amino acids 168-181, 184-203 and 200-212 of proendothelin-1.

5. The kit according to claim 1 or 2, characterized in that it is designed as a kit for performing immunochromatographic bedside tests.

6. The kit according to claim 1 or 2, characterized in that the antibody is an affinity-purified polyclonal antibody.

7. The kit according to claim 1 or 2, characterized in that said antibody is obtained by immunizing an animal with an antigen comprising a sequence selected from the peptides SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6.

8. A kit according to claim 1 or 2, characterized in that it comprises two different antibodies, one of which is labeled and the other of which is or can be selectively bound to a solid phase.

9. Kit according to claim 1 or 2, characterized in that it comprises two different antibodies, both antibodies being present in a liquid reaction mixture in a dispersed manner, wherein the first labeling component of the labeling system binding to the first antibody is part of a fluorescence or chemiluminescence based quenching or enhancing labeling system and the second labeling component of the labeling system binds to the second antibody, such that after binding of both antibodies to the peptide fragment to be detected, a measurable signal is generated which enables detection of the sandwich complex formed in the measurement solution.

10. Kit according to claim 9, characterized in that the labeling system comprises a cryptate or chelate of a rare earth element and a fluorescent or chemiluminescent dye.

11. A polyclonal antibody which specifically binds to a polypeptide consisting of amino acids SEQ ID NO: 4. 184-and 203-amino acid SEQ ID NO: 6 and 200-212 amino acids SEQ ID NO: 5 amino acid sequence of amino acid.

12. Antibody according to claim 11, characterized in that it is an affinity-purified polyclonal antibody.

13. Kit according to claim 1 or 2, characterized in that it comprises at least: (a) a first antibody according to any one of claims 11 and 12, (b) a second further antibody according to any one of claims 11 and 12, wherein one antibody is labelled and the other antibody is immobilised or immobilised, and (c) a standard peptide having an amino acid sequence comprising at least amino acids 168-203 or 168-212 of proendothelin.

14. Kit according to claim 13, characterized in that the immobilized antibodies are present in a form immobilized on the wall of the test tubule.