Patent Application
20080026414
The investigations carried out and the most significant results of the investigations are explained more exactly below, reference being made to figures. In the figures:
a shows the measurement of plasmas of 5 groups of human patients with different diseases/diagnoses, compared with the measurements for apparently healthy persons; the dotted line indicates the maximum value found in healthy persons (line for 100% specificity, based on healthy controls);
b shows a diagram corresponding to
The method according to the invention relates in its most general aspect to the determination of a relatively long-lived peptide fragment of proendothelin-1 which does not contain the amino acid sequences of endothelin-1 or its precursor big endothelin, in whole blood, plasma or serum samples, i.e. in the circulation of patients, for the indirect determination of the formation of endothelins, in particular of endothelin-1, in serious diseases. According to a preferred embodiment, the peptide fragment determined is a C-terminal fragment to which two antibodies bind which bind to peptides having amino acid sequences which correspond to the positions 168-181 and 200-212 of preproendothelin-1.
For the practical implementation of the invention, noncompetitive sandwich assays, for example of the type as used for the more far-reaching detailed investigations and described more exactly below, are particularly preferably provided.
Compared with competitive immunoassays, noncompetitive sandwich immunoassays (two-sided immunoassays) have a number of advantages, which include the fact that they can be better designed than solid-phase assays (heterogeneous assays), may be more rugged in terms of handling, can give measured results with a higher sensitivity and are also more suitable for automation and series measurement. Moreover, they can also provide additional information compared with competitive immunoassays which operate with only one type of antibody, in that sandwich immunoassays recognize only those molecules or peptides with which both binding sites for the antibodies used in the sandwich formation are present on the same molecule.
The antibodies which may be used may in principle be any desired suitable monoclonal and/or polyclonal antibodies, but affinity-purified polyclonal antibodies are currently preferred.
Particularly preferably, the antibodies are obtained by immunization of an animal, in particular of a sheep, with an antigen which contains a synthetic peptide sequence which corresponds to a short amino acid sequence of preproendothelin-1 and an additional cysteine residue at the N-terminus. In the following experimental section, in particular antibodies which bind to the amino acid sequences 161-181 and 200-212, and their use in an assay are described. However, in the course of the investigations, additional antibodies which accordingly bind to the positions 184-203 and 136-148 were also used. The additional results obtained with these further antibodies in measurements are discussed only globally in this Application.
In a preferred embodiment, the method is carried out as a heterogeneous sandwich immunoassay, in which one of the antibodies is immobilized on any desired solid phase, for example the walls of coated test tubes (e.g. of polystyrene; “coated tubes”; CT) or on microtiter plates, for example of polystyrene, or on particles, for example magnetic particles, while the other antibody carries a residue which represents a directly detectable label or permits selective linkage to a label and serves for detecting the sandwich structures formed. Delayed or subsequent immobilization with the use of suitable solid phases is also possible.
In principle, all marking techniques which can be used in assays of the type described may be employed, including marking with radioisotopes, enzymes, fluorescent, chemoluminescent or bioilluminescent labels and directly optically detectable color markers, such as, for example, gold atoms and dye particles, as are used, in particular for so-called point-of-care (POC) or accelerated tests for determination in whole blood samples. In the case of heterogeneous sandwich immunoassays, the two antibodies may also have parts of a detection system of the type described below in relation to homogeneous assays.
It is therefore within the scope of the present invention also to design the method according to the invention as an accelerated test.
The method according to the invention can furthermore be designed as a homogeneous method in which the sandwich complexes formed from the two antibodies and the peptide fragment to be detected remain suspended in the liquid phase. In such a case, it is preferable to mark both antibodies with parts of a detection system which permits signal generation or signal triggering when both antibodies are integrated into a single sandwich. Such techniques can be designed in particular as fluorescence amplification or fluorescence extinction assays. A particularly preferred method of this type relates to the use of detection reagents to be used in pairs, as described, for example, in U.S. Pat. No. 4,822,733, EP-B1-180 492 or EP-B1-539 477 and the prior art cited therein. They permit a measurement which selectively detects only reaction products which contain both marking components in a single immune complex, directly in the reaction mixture. The technology which is available under the brands TRACE® (Time Resolved Amplified Cryptate Emission) and KRYPTOR® and which implements the teachings of the above-mentioned application may be referred to as an example.
In the investigations by the Applicant, it was found that the determination, according to the invention, of the C-terminal peptide fragment of preproendothelin-1 gives highly interesting and relevant results. As will be shown below, this statement applies not only to the sepsis diagnosis but also to cardiac diagnosis and cancer diagnosis.
It is furthermore assumed that the assays according to the invention can also be particularly advantageously carried out as part of a so-called multiparameter diagnosis, in particular both in the area of cardiac diagnosis and in sepsis and cancer diagnosis. Further parameters determined thereby are, for example, the cardiac parameters ANP, BNP, proANP, proADM and proBNP or sepsis parameters which are selected, for example, from the group which consists of anti-ganglioside antibodies, the proteins procalcitonin, CA 125, CA 19-9, S100B, S100A proteins, LASP-1, soluble cytokeratin fragments, in particular CYFRA 21, TPS and/or soluble cytokeratin-1 fragments (sCY1F), the peptides inflammin and CHP, other peptide prohormones, glycine-N-acyltransferase (GNAT), carbamoylphosphate synthetase 1 (CPS 1) and C-reactive protein (CRP) or fragments thereof. In said multiparameter assays, it is intended to determine the measured results for a plurality of parameters simultaneously or in parallel and to evaluate them, for example, with the aid of a computer program which also uses diagnostically significant parameter correlations.
The invention is explained in more detail below by a description of the preparation of the preferred assay components, the procedure of a preferred embodiment of an assay of the sandwich type and the results of the determination of a C-terminal peptide fragment in EDTA plasmas of control persons and of sepsis, cardiac and cancer patients, obtained with the use of such an assay.
1. Peptide Syntheses
Derived from the known amino acid sequence of human preproendothelin-1 (SEQ ID NO:1), three ranges were selected (Pos. 168-181, 184-203, 200-212). In each case supplemented by an N-terminal cysteine residue, these ranges were chemically synthesized as soluble peptides by standard methods, purified, quality-controlled by means of mass spectrometry and reversed phase HPLC and lyophilized in aliquots (JERINI AG, Berlin, Germany). The amino acid sequences of the peptides are:
Furthermore, the following peptide was synthesized as a standard for calibrating the assays:
2. Conjugation and Immunization
The peptides PCT15 and PCW14 were conjugated with the carrier protein KLH (keyhole limpet hemocyanine) by means of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) (cf. operating instructions “NHS-Esters-Maleimide Crosslinkers”, from PIERCE, Rockford, Ill., USA). Sheep were immunized with these conjugates according to the following scheme: each sheep initially received 100 μg of conjugate (stated mass based on the peptide fraction of the conjugate) and then 50 μg portions of conjugate every 4 weeks (stated mass based on the peptide fraction of the conjugate). Beginning with the fourth month after beginning of the immunization, 700 ml of blood per sheep were taken every 4 weeks and antiserum was obtained therefrom by centrifuging. Conjugations, immunizations and recovery of antisera were carried out by MicroPharm, Carmarthenshire, UK.
3. Purification of the Antibodies
The peptide-specific antibodies were prepared in a one-step method from the antisera which had been recovered beginning with the fourth month after immunization.
For this purpose, the peptides PCT15 and PCW14 were first coupled to SulfoLink Gel (cf. operating instruction “SulfoLink Kit”, from PIERCE, Rockford, Ill., USA). In each case 5 mg of peptide per 5 ml of gel were offered for coupling.
The affinity purification of peptide-specific antibodies from sheep antisera against both peptides was carried out as follows:
The peptide columns were first washed three times alternately with 10 ml each of 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 the antisera were filtered with 0.2 μm, and the column material present was added. For this purpose, the gel was quantitatively rinsed from the column with 10 ml of binding buffer. The incubation was effected overnight at room temperature with swirling. The batches were transferred quantitatively into empty columns (NAP 25, Pharmacia, emptied). The runnings were discarded. The columns were then washed protein-free with 250 ml of binding buffer (protein content of the wash eluate <0.02 A280 nm). Elution buffer was added to the washed columns, and 1 ml fractions were collected. The protein content of each fraction was determined by means of the BCA method (cf. operating instructions of PIERCE, Rockford, Ill., USA). Fractions having protein concentrations >0.8 mg/ml were pooled. After protein determination of the pools by means of the BCA method, yields of 97 mg for the anti-PCT15 antibody 0407-pAk and 60 mg for the anti-PCW14 0410-pAk antibody were obtained.
4. Marking
The anti-PCW14 0410-pAk antibody was treated as follows:
500 μl of the purified antibody were rebuffered in 1 ml of 100 mM potassium phosphate buffer (pH 8.0) according to the operating instructions over an NAP-5 gel filtration column (Pharmacia). The protein concentration determination of the antibody solution gave a value of 1.5 mg/ml.
For chemiluminescence marking of the antibody, 10 μl of MA70 acridinium-NHS-ester (1 mg/ml; from HOECHST Behring) were added to 67 μl of the antibody solution and incubated for 15 minutes at room temperature. Thereafter, 423 μl of 1 M glycine were added and incubation was effected for a further 10 minutes. Thereafter, the marking batch was rebuffered according to operating instructions over an NAP-5 gel filtration column (Pharmacia) in 1 ml of mobile phase A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4) and freed from low molecular weight constituents. A gel filtration HPLC was carried out for separating off final residues of labels not bound to antibodies (column: Waters Protein Pak SW300). The sample was applied and was chromatographed at a flow rate of 1 ml/min with mobile phase A. The wavelengths 280 nm and 368 nm were measured using a flow photometer. The absorption ratio 368 nm/280 nm as a measure of the degree of marking of the antibody was 0.10 at the peak. The fractions containing monomeric antibodies (retention time 8-10 min) were collected, and were collected in 3 ml of 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% sodium azide, pH 7.4.
5. Coupling
The anti-PCT15 antibody 0407-pAk was treated as follows:
Irradiated 5 ml polystyrene tubes (from Greiner) were coated with purified antibody as follows: the antibody was diluted to a concentration of 6.6 μg/ml in 50 mM Tris, 100 mM NaCl, pH 7.8. 300 μl of this solution were pipetted into each tube. The tubes were incubated for 20 hours at 22° C. The solution was filtered with suction. Each tube was then filled with 4.2 ml of 10 mM sodium phosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5. After 20 hours, the solution was filtered with suction. Finally, the tubes were dried in a vacuum drier.
An assay buffer of the following composition was prepared: 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin (BSA), 0.1% unspecified sheep IgG, 0.1% sodium azide, pH 7.4
The above-mentioned chemically synthesized peptide (peptide PSW44) which corresponds to the positions 169-212 of preproendothelin-1 serves as standard material. This was serially diluted in horse normal serum (from SIGMA). Concentrations according to the weight of the peptide taken were ascribed to the standards thus prepared.
Measuring samples were EDTA plasmas of apparently healthy persons, of patients with sepsis and of patients with various cardiovascular diseases.
50 μl of standards or samples and 200 μl of assay buffer were pipetted into the test tubes. Incubation was effected for two hours at 22° C. with shaking. Thereafter, washing was effected 4 times with 1 ml of wash solution (0.1% Tween 20) each time per tube and the latter were allowed to drip off. 200 μl of assay buffer, containing 1 million RLU (relative light units) of the MA70-marked antibody, were then pipetted. Incubation was effected for two hours at 22° C. with shaking. Thereafter, washing was effected 4 times with 1 ml of wash solution (0.1% Tween 20) each time per tube, the latter were allowed to drip off and the chemiluminescence bound to the tube was measured in a luminometer (from BERTHOLD, LB952T; base reagents BRAHMS AG).
Using the MultiCalc software (spline fit), the concentrations of the samples were read from the standard curve.
The analyte measurable using the sandwich immunoassay developed (antibody against the positions 168-181 and 200-212) is referred to below as C-terminal proendothelin or Ct-proendothelin. A typical standard curve for the test developed is shown in
In order to examine the question as to whether problems were to be expected in a measurement of the C-terminal peptide fragment owing to insufficient stability in a sample or measuring solution, 5 sepsis plasmas were measured in each case fresh and after storage for 12 hours at room temperature. The results are summarized in
By means of the test, plasmas of cardiological and sepsis patients were measured. The results obtained are shown in
When the samples were measured using a modified assay in which one of the antibodies of the above-mentioned sandwich assay was replaced by an antibody which detected the amino acids 184-203 of preproendothelin-1, substantially identical results were obtained, as expected.
On the other hand, when one of the antibodies used recognized an amino acid sequence which is localized more closely to the N-terminus of the preproendothelin (32-52 or 136-148), it was not possible to obtain measured values raised compared with healthy persons. This indicates that proendothelin as such was not present in the plasma samples measured and is not proteolytically processed only with formation of big endothelin, but that the C-terminal sequence 93-212 liberated is also further cleaved, it being necessary for at least one such cleavage point to be present in the range of the amino acids 149-167. The statement applies to the plasmas of patients with the diseases investigated. However, it cannot be ruled out that, for example, the entire C-terminal fragment 93-212 is retained in other patient groups and its selective measurement can deliver diagnostically relevant results.
| Number | Date | Country | Kind |
|---|---|---|---|
| 04003295.5 | Feb 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/01359 | 2/10/2005 | WO | 00 | 5/23/2007 |
