DETECTION OF CARDIAC MUSCLE NECROSIS BY IMMUNOASSAY AND APPROPRIATE ANTIBODIES THEREFOR

Abstract: The invention relates to a diagnostic method for determination of cardiac muscle necrosis by measuring the level of cardiac specific troponin I-C complex in blood using an immunoassay. The method is characterized by (1) incubating plasma or serum sample with a primary antibody specific to troponing I, C, or I-C; (2) separation of the formed immunocomplex involving the primary antibody and troponin I-C; and (3) the measurement of the amount of the isolated immunocomplex with a second labelled antibody (anti-troponing I, anti-troponing C or anti-troponing I-C), so that possible contribution from separate I or C alone is not measured; or alternatively, when the primary antibody is specific only to I-C the formed immunocomplex can be measured also by labelled I-C in a competitive manner.

Description: DETECTION OF CARDIAC MUSCLE NECROSIS BY IMMUNOASSAY AND APPROPRIATE ANTIBODIES THEREFORE FIELD OF INVENTION This invention relates to clinical diagnostics and more particularly to the diagnostics and therapy follow-up of heart muscle necrosis. The invention concerns with a new approach to monitor the condition of heart by leakage of two proteins, troponin I and C, from necrotic heart muscle. BACKGROUND OF THE INVENTION Heart infarction is one of the commonest causes of death of adult persons in developed countries. In case there is available quick diagnosis after infarction followed by appropriate medicinal treatment or surgical operation, a significant part of the patients can be rescued. The required laboratory diagnostics is traditionally based on the detection of elevated enzyme activities of e.g. creatinine kinase (CK) , lactate dehydrogenase (LD) , or aspartate a inotran erase (GOT) in serum. The drawback is, however, the ambiguousness of interpreting the results since the enzymes can also originate from organs other than heart. Hence, the diagnostics of the acute myocardial infarction (AMI) is to be based on the laboratory tests accompanied with rather diffuse clinical symptoms or on electrocardiography (ECG). To achieve reliable diagnosis of heart infarction, the nonspecificity of the customary laboratory tests brings about the need for a few sequential samples as a function of time. This results in a slowness of getting the final diagnosis that is very severe drawback for the required therapeutic actions (for a review, see Journal of Clinical Immunoassay, Vol. 17, No.l, pp.6-48). An objective for future is to diagnose " infarction by one serum sample only. This would require a reliable marker which can be measured in high accuracy. The resulting value can be interpreted statistically to reconstruct the history of the infarction. The building block proteins of the heart muscle contractile apparatus have been subject of intensive studies for their applicability to the heart infarction diagnostics (F.C. Apple: Laboratory Medicine, May 1992, Vol. 23, No. 5, pp. 311-317). The troponins are proteins located on the thin film filament of the muscle contractile apparatus. There are three troponins (Tns), troponin T (tropomyosin-binding subunit with molecular mass of 39kD), Tn I (actomyosin- adenosine triphosphatase-inhibiting subunit with molecular mass of 26.5kD), and Tn C (calcium-binding subunit with molecular mass of 18kD), acting together to regulate the muscle contraction. The heart Tns, excluding Tn C, are immunologically different from those in skeletal muscle. This forms the grounds of utilizing troponins as specific markers of heart muscle damages. Recently some diagnostic kits for heart Tns I and T have become commercially available. It has been thought that cardiac Tn C is not suitable for heart diagnostics at all since the slow-twitch skeletal isoform of Tn C is identical to the cardiac isoform. Tn I also exists in cardiac, fast- twitch and slow-twitch skeletal isoforms. The cardiac isoform of Tn I is about 40% dissimilar from the skeletal isoform and contains an additional specific functional amino acid sequence on the N-terminus. Despite of the instability of Tn I, it is considered as such one of the most promising markers of the cardiac injury (SCRIPPS NEWS, CA, USA, 1993, Vol. 7, No.2.). SUMMARY OF INVENTION The object of this invention is an improved method for determination of the status of cardiac muscle necrosis by immunoassay comprising of simultaneous determination of two cardiac muscle troponins I and C (Tn I and Tn C), able to bind to each other to form a complex I-C. The invention exploits the observation that variable portion of Tn I in serum is complexed with Tn C. The complexed Tn I (I-C) and Tn I - complexable to externally added Tn C (total Tn I-C), or the ratio Tn I-C/total Tn I-C, can be used as an improved measure for heart infarction diagnostics. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES Figure 1; Gel chromatography of Tn I added to a normal serum and of a serum of a patient with acute myocardial infarction (AMI) on a column separating proteins according to their molecular masses from 10,000 to 1 million daltons (calibration points with known protein standards indicated) . Figure 2: Gel electrophoresis pattern of Tns C, I, and T on gradient polyacrylamide gel (10-20% acrylamide). Figure 2 shows that Tn I exists in serum of AMI patients in a complex mainly with Tn C. Figure 3; Analysis of normal serum and three sera from suspected AMI patients (AMI Sample 1-3) by using different monoclonal antibodies. NHS refers to Normal Human Serum. Figure 4 : Figure 4 shows that when pure Tn I is added to normal serum (all-male serum) the signal increases indicating that normal serum pool contains Tn C. External Tn C increases signal dramatically whenever Tn C is detected with a labeled Tn C-specific antibody (here clone 1A2). SA refers to Streptavidin coated onto a microtiter plate surface and B to Biotin. Figure 5: Mesurement of Tn I-C complex in sera of 11 potential AMI patient after hospital admission by a sandwich assay (coating antibody 8E10 and labeled antibody 1A2). Black bars refer to Tn I-C and grey bars to total Tn I-C complex obtained by adding Tn C (1 μg/microtiter plate well) . Figure 6; Correlation between Tn I and Tn I-C complex (10F4 coating and 7F5 labeling for Tn I; 8E10 coating and 1A2 labeling for Tn C) in sera of potential AMI patients sampled in different times from the infarction. The values of the large squares are not included in the regression line. They represent exceptional values in the very beginning and the late stage of infarction. Figure 7: Variation of signal from Tn I (10F4 coating, 7F5 labeled) and Tn I-C (8E10 coating, 1A2 labeled) as a function of time in sera of AMI patients. Figure 8; Signal obtained out of Tn I-C disassembled to obtain Tn I by addition of EDTA (antibodies 10F4 and 1A12) (Example 2) Table I: Correlation coefficient between measurement of troponin I (by methods II and 12), Tn I-C, total Tn I-C, CK, and CKB. BROAD DESCRIPTION OF THE INVENTION Troponins T, I, and C are immobilized together as a group in the muscle contractile apparatus. Troponin T is found to contain an unbound cytosolic pool of 6% of total myocardial Tn T content. Washout of this pool from muscle cells may cause the peak of Tn T concentrations in serum on the first day of early reperfusion of the infarct zone. There is a rapid disappearance of Tn I from myofibrils during myocardial infarction but Tn T was shown to persist in the zone nearly unaltered for six days. The degradation rate of Tn T is comparable to that of myosin light chains. The difference in the disappearances of Tns I and T from myofibrils may account for the differences in the release kinetics of both markers in patients with nonperfused myocardial infarction. The continuous degradation of myofibrils during infarct evolution and healing results in a resistant egress of myofibril components into circulation (Katus, H.A., et al. in Laboratory Medicine, Vol. 23, 1992, p.312) . As stated above, the release kinetics of Tns into serum from necrotic zone of heart is complicated. Tn C is not considered useful as the marker of myocardial infarction, whereas Tn I is for a long time considered as one of the most promising specific markers. According to this invention a significant, but variable, portion of Tn I in serum of AMI patients exists as a complex of Tn I with Tn C (Tn I-C) bound with other proteins and this Tn I-C is used for heart infarction diagnostics either as such or so that external Tn C is added (total I-C). Additional advantages are obtained from concomitant stabilization of Tn I by Tn C and in this way Tn I content in serum is standardized resulting in a more accurate and reproducible measure for the infarction. Scheme 1 illustrates the principal reactions which the present invention is based onto. According to their biological function, the molecular conformations of each Tns and their mutual interaction change considerably during the contraction and release of muscle tension. Concomitantly the immunological properties of Tns and especially that of Tn I shall be different when it is free or bound to Tn C. If the sample contains both free Tn I and complex Tn I-C, the primary antibody binds different portion of free Tn I and Tn I-C complex. It is shown in this invention that individual patient samples contain f irly different ratios of free and complexed Tn I. Thus, the measurement of only Tn I does not necessarily probe the real amount of Tn I. If the second labeled antibody also binds to Tn I, the complication arising from changed specificities along with conformational changes is still amplified. A further difficulty originates from the fact that free Tn I contains hydrophobic areas in the molecule that cause its strong nonspecific binding to various materials including glass and plastics. This area locates most probably at the Tn I-C interface or the Tn I-C interaction otherwise affects occurrence of such areas. Since different serum samples contain different ratio of Tn I-C/ total Tn I-C contents, and since the determination of exact nonspecific binding background in the signal is tedious, it is highly desirable to stabilize the serum samples in the respect of these background effects. In conclusion, the ratio Tn I-C /total Tn I-C includes considerably more information about the history and degree of damage of the heart infarction than measurement of Tn I. Pure added Tn I is very unstable molecule in serum and in other solutions. Its immunological activity decreases to 20% within one hour at 37°C. British Patent Application GB 2 275 774 A claims that Tn I standard preparation for immunoassays can be stabilized by addition of Tn C and optionally Mg ++ - or Ca + -ions. However, the cited invention did not exploit the stabilizing effect of Tn I by Tn C being released from heart, skeletal muscle, or added from an external source. In the present invention a related stabilization as with the cited Tn I standard preparation above, is exploited for increasing the reproducibility and sensitivity of Tn I assays. Such external Tn C can originate from any source including those produced in microbes by the recombinant techniques. The stabilization also provides an additional advantage since the clinical samples may be stored for an undetermined periods before the measurement. Such stabilization can be carried out by adding Tn C into blood sampling tube, to serum, or to a sample to be analyzed. DETAILED DESCRIPTION OF THE INVENTION The antibodies mentioned in this invention refer to those found in the catalogs of Hytest Ltd., Turku, Finland or BioSpacific Ltd, California, USA. In the present invention, Fast Protein Liquid Chromatography (FPLC) technique with a gel chromatography column having molecular mass separation range of lOkD- 1000 kD was applied in non-denaturating conditions (Figure 1). The profile of proteins as measured by absorbance at 280 nm in AMI patient samples does not differ from normal serums; three major peaks with molecular masses of about 65 kD, 160 kD and 1000 kD appeared with both (not shown in Figure 1) . Figure 1 shows that any of the Tns does not exist in the serum in free form but are bound to other biomolecules and usually separated into two fractions in the chromatographic conditions. Also pure Tn I added to normal serum, not containing heart-originated proteins, rapidly form the aggregates . Figure 2 shows that when Tns I, C, and T are captured by an immunoaffinity adsorbent from serum pool of AMI patients, followed by washing and eluting out of the adsorbent at low pH, only a complex containing of Tns I and C are detected. Thus Tn T does not significantly bind to Tn I-C. Figure 2 shows pure standard Tns, I, C, and T specifically detected on an electrophoresis gel. The three lanes left side represents electrophoresis of a sample of a serum from a pool of AMI patients also specifically detected by Tn I, Tn C, and Tn T -specific antibodies. The serum from AMI patients was first applied on an immunoaffinity column of Sepharose 4 B CL derivatized with monoclonal antibody known to bind the complex Tn " T-I-C. This fraction was eluted from the column and subjected to the electrophoresis followed by the Western blotting. The membrane was allowed to react with specific mice antibodies against each individual Tn forms and stained thereafter with goat anti-mouse IgG labeled with horseradish peroxidase which was detected by chemiluminescence. The bands at the molecular masses at 55 and 25 kD originate from the heavy and light chains of the mouse antibodies used to capture the complex Tn I-C-T being partly detached from the affinity column by the acid eluent. Figure 3 characterizes the test system of the present invention by different antibodies. The secondary labeled antibody (7F5) was specific to Tn I. The low signals with anti-Tn T antibody (1C11) as the primary catching antibody indicates that Tn T is not involved in complex with Tn I whereas anti-Tn C antibodies (1A2 and 7E4) yield remarkable signals. The antibody 10F4 is not strictly specific to Tn I but preferably recognizes Tn I in complex I-C. Figure 4 shows that serum pool of healthy blood donator men (all-male serum) contains Tn C which can be found and quantitated in the serum by adding Tn I purified from human heart. This proves that Tn C itself is a poor marker of heart muscle damages. If the catching antibody for Tn I is omitted in the test system, the background signals from the secondary, labeled antibody (1A2 specific to Tn C) are small and are even decreased by adding Tn I in concentration of 30 ng/ml (black bar in Figure 4 b). If Tn C is added into the same system, the background due to adsorption of Tn C onto the microtiter plate surface is slightly increased (grey bar in Figure 4 b) while addition of Tn I decreases it (black bar). In Figure 4 c, Tn I catching antibody is immobilized and pure Tn I (30 ng/ml) is added into normal serum (black bar). Since the background (grey bar) is small, and the other background controls are valid (see before), the normal serum contains Tn C If a large excess of Tn C is added into the system similar to Figure 4 c, the signal increase (black bar) is dramatic while the background keeps still relatively small (Figure 4 d) . As is generally known in the immunological test systems related to those of experiment of Figure 4 d, addition of Tn C in increasing amounts, will increase the signal only to a certain point and thereafter the signal will decrease due to the so-called hook-effect. It can be avoided by employing sequential incubations with Tn C and secondary antibody with washing the wells between the incubations. Figure 5 exemplifies the effects of externally added Tn C into serum samples of 11 potential AMI patients with high CK values . In all cases the signal increased by adding Tn C into the system being attributable to the additional formation of complex Tn I-C. The increase is not constant but considerably varies from patient to patient. The signal without addition of Tn C (Tn I-C complex in the serum; grey bars) compared to the signals after adding Tn C in excess (total Tn I-C, black bars) are around 75% in the average. This value can change accordingly with the experimental conditions. Free Tn C originating from skeletal muscle may interfere with the values obtained for Tn I-C complex from heart in the system similar to Figure 5, where Tn I is captured by heart-specific antibody. The situation can be illustrated by two patients, one having heart attack after physical exercise and the other one without it. Both are assumed to have equal size of the necrotic zone. After the physical exercise free Tn C leaked from skeletal muscles, shall stabilize the Tn I leaking from heart and thus the increase by addition of external Tn C into such serum will be less than with the other one. On the contrary, the patient withcut Tn C leakage from skeletal muscle may have lower levels of measurable Tn I since it can have been reversibly denatured in the lower " concentrations of stabilizing Tn C leaking from heart muscle only. However, in the latter case the signal increase within adding external Tn C can be considerably higher. Hence, the determination pf Tn I-C and total Tn I-C values can yield extra information about the condition of the patient. Table 1 shows results obtained by statistical treatment of the correlation coefficients of diagnostic results randomly chosen among AMI patients taken into hospital. It shows that correlation coefficients are usually highest in the cases the total Tn I-C, or when complex Tn I-C, or when Tn I-C (marked I+IC in Table 1) is measured by two labeled antibodies, one preferably binding to Tn C and the other one to Tn I. Total Tn I-C complex shows the best correlation to CK and CKB while only poor correlation occurs with the methods where CK and CKB are compared with Tn I measured by two different sandwich assays with two antibodies against Tn I (marked II : 7F5 coating, 10F4 labeled; and 12 : 8E10 coating, 7F5 labeled). Figure 6 shows that generally there is a correlation between Tn I and complex Tn I-C but at early and late stages after hospital admission there appear large variations from the regression line. It is noticeable that this effect can be utilized to evaluate the history of infarction. Figure 7 illustrates further the advantages of this invention by showing that the time-courses of the increase of the signal from Tn I (dashed line with using antibodies 10F4, 7F5) and complex Tn I-C (solid line with using antibodies 8E10, 1A2) are related in form but the signal from Tn I-C is much higher thus providing considerably higher sensitivity of the assay or earlier diagnosis. Figure 8 shows that addition EDTA increases the signal when the complex Tn I-C is detected by antibody pairs 10F4 and 1A12 (coating and labeled, respectively). Since the antibody 1A12 binds to Tn I and the signal from it increases by adding EDTA, the epitope of this antibody must situate near the mutual binding site of Tn I and Tn C. This provides another route to measure the total Tn I-C. An additional advantage of the present invention is achieved by measuring the ratio of the complex Tn I-C in the presence or absence of EDTA or another complexing agent such as EGTA, DTPA, or citrate. Such treatment will destroy the Tn I-C complex as described in the British Patent Application GB 2200 358 A. In case the catching antibody is specific to Tn I, it is possible to measure the free and Tn I-C complex - releasable Tn C. If the secondary antibody has the same binding site as Tn C, it will measure free Tn I or Tn I unveiled from complex Tn I-C by EDTA. Although apparently the system described above allows to measure the ratio of free Tn I / Tn I-C complex, there remains a drawback of nonspecific binding of Tn I to majority of materials used in the normal assays, including glass and plastics. It is, however, presumable that suitable materials can be found which do not absorb Tn I in nonspecific manner by selecting other materials or by presaturating normally used polystyrene or glass with appropriate compounds like proteins, polyvinylalcohol, polyvinylpyrrolidone, chitosan, carboxymethyl cellulose, gelatinized starch, or other related material. The main embodiment of the present invention is that while Tn C is not useful for heart infarction diagnostics for its unspecificity, as such, it can be exploited indirectly for such diagnostics in the following ways: 1) by exploiting the observation that a considerable, but largely variable, portion of Tn I exists as a complex with Tn C in serum of AMI patients and by utilizing the ratio Tn I-C / total Tn I-C or Tn I-C - complexable with Tn C as a measure for heart muscle necrosis, 2) by stabilizing Tn I from denaturation with an external Tn C added into the sample, 3) by standardizing the signal/background ratio in different serum samples by driving Tn I totally to the stable complex Tn I-C, 4) by measuring the ratio Tn I-C/ Tn C - detachable from the complex Tn I-C, by measuring Tn I with and without addition of a strong complexing agent, such as EDTA in the serum sample, 5) by measuring simultaneously Tn I-C and total Tn I-C in such a manner that Tn I-C is measured by a specific antibody with label 1, and total Tn I-C is measured by a second antibody bound with troponin C and labeled with another label 2. Alternatively Troponin C is labeled with label 2. Labels 1 and 2 are preferably measured simultaneously at different wavelengths enabling to get the ratio Tn I-C / total Tn I-C in the same condition. Alternatively, the first and second labels can be other detectable molecule such as different enzymes, radioactive atoms, luminescence molecules, or fluorescing molecules with different wavelengths of detection. The complex Tn I-C can be also measured by a competitive assay where a labeled Tn I-C is added into a sample and a competition for the sample Tn I-C with the labeled Tn I-C is created. The binding antibody for the complex can be prepared from a polyclonal antibody through a proper fractionation procedure or by preparing a specific monoclonal antibody. While the experimental studies herein are performed with a time-resolved techniques using fluorescence from europium ion, they are not by no means limited to this special technique. Equally well similar diagnostic methods can be performed with other usually available methods like ELISA with using any enzyme label, luminescence, radioactive or any other detection known in the prior art. The invention is further illustrated by the following non¬ limiting examples. EXAMPLE 1 A time-resolved fluoroimmunoassay of Tn I-C and total Tn I-C a) Labeling of monoclonal antibodies with europium chelate N 1 -(p-isothiocyanatobenzyl)-diethylenetriamine-N 1 ,N 2 ,N 3 ,N 3 - tetraacetic acid europium complex (DTTA) (Wallac, Turku, Finland) in a 60-fold mplar excess is allowed to react with the appropriate antibody at pH 9.8 overnight. The labeled antibody is separated from excess of DTTA on a column of Sephadex G-50 (1x5 cm) and Sepharose 6 B (1x50 cm) by using 50 mmol/L Tris-HCl buffer, pH 7.8, containing 9 g/L of NaCl and 0.05 % NaN 3 as the eluting agent (TSA buffer). Bovine serum albumin (1 g/L) is used as stabilizer in the solution. b) Biotinylation of monoclonal antibodies The monoclonal antibody in concern (for example, 8E10) is allowed to react with 20-molar excess of an isothiocyanato- phenyl derivative of trioxyethylene-amidibiotin (SCN- biotin) in 50 mmol/L sodium carbonate buffer, pH 9.8, for 2 h at the room temperature. The final concentration of the antibody in 1 mL of reaction mixture shall be around 1 mg/mL (described in ref. Hemmila I, Heikkila J, Leivo P. Direct and indirect immunofluorometry of thyrotropin [Abstract 671].Clin Chem 1989; 35:1206). The biotinylated antibody is separated from the reaction mixture by gel filtration using NAP-10 and PD-10 disposable columns (Pharmacia, Uppsala, Sweden) using TSA buffer as the eluent. Bovine serum albumin (1 g/L) is used as stabilizer in the solution . c) Assay procedure The time-resolved assay of Tn I or Tn I-C is performed with the streptavidine-coated microtiter wells (Wallac, Turku, Finland). Total Tn I-C assay is performed essentially similarly as Tn I and Tn I-C assays . Patient serum samples (or standards prepared in normal human serum) are added in the volume of 50 μL in to the wells of microtiter. Biotinylated catching antibody (300 ng/well), Eu-labeled antibody 200 ng/well, and in the case of measurement of total Tn I-C, Tn C 50 -2000 ng/well are added in TSA buffer containing 0.1 % bovine serum albumin (Wallac, Turku, Finland). The final reaction volume is 100 μL. After incubation for 1 h with gentle agitation at the room temperature, the strip wells are washed six times with Wash solution and 200 μL of Enhancement solution/well (Wallac, Turku, Finland) are added. After 5 min incubation with shaking and after a further 10 min without shaking the fluorescence is measured with a 1234 Delfia Research Fluorometer (Wallac, Turku, Finland). EXAMPLE 2 Measurement of Tn I-C with and without EDTA Microtiter plates are coated by with an antibody (10F4, Hytest, Turku, Finland) in 50 mmol/l sodium carbonate buffer, pH 9.2, at a concentration of 10 μg /mL overnight. The plate wells are washed six times with a wash solution (Labmaster Ltd., Turku, Finland) and saturated with bovine serum albumin. The secondary antibody (1A12, BiosPacific, USA) is labeled with europium chelate according to the instructions from the manufacturer (Wallac Ltd., Turku, Finland). The assay is carried out by adding 25 μL serum to the coated well and then 75 μL of assay buffer (Labmaster Ltd.) containing either 10 mM EDTA or not. After one hour incubation the wells are washed six times with the wash solution (Labmaster Ltd., Turku, Finland) and then the labeled antibody (200 ng/well) is added in the volume of 100 μL in the assay buffer (Labmaster Ltd. ) . After 1 hour incubation the wells are washed six times with the washing solution and 200 μL of Enhancement Solution (Wallac Ltd., Turku, Finland) is added and the fluorescence is measured after 10 minutes with a gentle shaking of the plate. Ten serum samples of suspected AMI patients with elevated values of creatinine kinase B (from Central Hospital of the University of Turku) were analyzed. The average signal without EDTA was 96,000 cps and with EDTA 190,000 cps (Figure 8) . EXAMPLE 3 Measurement of Tn I-C by a competitive assay A polyclonal antibody raised against human Tns I and C complex in mice is purified by immunoaffinity. Tn C is immobilized into the cyanogen bromide activated agarose according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden) by using 1 mg of Tn C per ml of moist activated gel. Similar gel is prepared for Tn I. The crude antibody solution (1 mL ascites fluid) is eluted through both 1-mL columns in buffered (pH 7.8) physiological salt solution. The proteins eluted through the both columns are adsorbed onto an immobilized Tn I where Tn C is allowed to complex (Tn I-C column). The protein obtained from Tn I-C column is coated onto a microtiter plate by passive adsorption (Tienhaara et al., Clin.Chem. (1990), 36/11, 1961) (100 μg/well, overnight) and the wells are washed six times with a Wash solution (Wallac Ltd., Turku, Finland). Tn I-C complex is labeled with europium chelate (Wallac Ltd., Turku, Finland) as the antibody in Example 1. Sample with a constant known amount of labeled Tn I-C are added in the assay buffer (Wallac Ltd., Turku, Finland) into the microtiter wells as in Example 1. The incubations, washings, and measurements are thereafter performed essentially as in Example 1. The content of Tn I-C is inversely proportional to the signal obtained from labeled Tn I-C. EXAMPLE 4 Measurement of Tn I-C and total Tn I-C by a double labeling technique. The assay of Tn I-C is performed as described in Example 1 until the second labeled antibody specific to Tn C (e.g. 1A2) is added into the reaction mixture and incubated and washed six times. After this procedure a new reaction is carried out in the same wells within the reaction mixture with another labeled antibody, such the antibody 7F5 which binds to another epitope than the primary antibody on Tn I, or Tn C is labeled with another lanthanide chelate than europium; such as samarium, terbium, or yttrium. This labeling reaction can be performed essentially as the one with the europium chelate as described in Example 1 or in reference, Pettersson et al., (1992) Clin.Chem. 38/10, 2038-2043. Alternatively, both labeled antibodies are added in the same time and measured with a time-resolved fluorometer (wallac Ltd., Turku, Finland) within the Enhancement solution by fluorescence at the different wavelengths from the separate metal ions. 18

Claims: CLAIMS 1. A diagnostic method for determination of cardiac muscle necrosis by measuring the level of cardiac specific troponin I-C complex in blood using an immunoassay characterized by (1) incubating plasma or serum sample with a primary antibody specific to troponin I, C, or I-C, (2) separation of the formed immunocomplex involving the primary antibody and troponin I-C, and (3) the measurement of the amount of the isolated immunocomplex with a second labeled antibody (anti-troponin 1. anti-troponin C or anti-troponin I-C), so that possible contribution from separate I or C alone are not measured; or alternatively, when the primary antibody is specific only to I-C the formed immunocomplex can be measured also by labeled I-C in a competitive manner. 2. The diagnostic method according to claim 1, characterized by a second measurement of troponin I-C complex comprising of measurement of total troponin I-C formed by externally added troponin C in amounts of 1-300 -fold molar excess to troponin I in patient plasma or serum sample containing cardiac troponin I. 3. The method according to claim 1 or claim 2 characterized by evaluation of the cardiac muscle necrosis by the ratio of signal with and without addition of troponin C. 4. A diagnostic procedure for the measurement of heart muscle necrosis in human blood characterized by the determination of the ratio in accordance with claim 3 using the immunochemical method described in claim 1 except that two different antibodies against troponin I are used in such a way that one of these antibodies has epitope on troponin I at the binding site of troponin C and the other one binds to any other epitope in troponin I, while the measurement is accomplished in the presence and absence of a chelating substance such as EDTA in concentration of 1-50 mM in the serum sample. 5. A diagnostic procedure for the measurement of heart muscle necrosis in human blood characterized by the determination of troponin I in human plasma or serum using a previous known method, and troponin I-C complex according to claim 1 or 2 and the ratio is utilized to estimate the degree of heart necrosis. 6. The diagnostic method of according to claim 2 characterized in that troponin C originates from human or animal sources, or is produced by recombinant techniques. 7. The diagnostic method according to claim 2 characterized by utilizing an antibody raised against unnatural troponin C, produced for example, by recombinant techniques in such a way that troponin C is able to complex with troponin I. 8. A diagnostic procedure for the measurement of the heart muscle necrosis in human blood, characterized by simultaneous immunochemical measurement of cardiac troponin I-C complex and cardiac troponin I complexable to troponin C in human plasma or serum by using dual labeling of detecting antibodies, or one antibody labeled with a detectable molecule and the added external troponin C labeled with another detectable molecule. 9. The method according to claim 1 characterized by the measurement of the signal by a time-resolved fluorescence using single or double labeling method according to claim 8. 10. The diagnostic method according to claim 2 characterized by the addition of troponin C into the sample into the concentration from 1 microgram to 1 milligram per ml of blood during taking the blood from patient, or alternatively the troponin C is placed into the sampling tube before, or troponin C is added later to the separated serum.

Inventors: ESKOLA JARKKO, KATROUKHA ALEXEI, KORPELA TIMO, SEVERIN EUGENII, SEVERIN SERGEY, ESKOLA, JARKKO, KATROUKHA, ALEXEI, KORPELA, TIMO, SEVERIN, EUGENII, SEVERIN, SERGEY
Applicants: ESKOLA JARKKO, FINNISH RUSSIAN JOINT BIOTECHN, KATROUKHA ALEXEI, KORPELA TIMO, SEVERIN EUGENII, SEVERIN SERGEY, ESKOLA, JARKKO, FINNISH-RUSSIAN JOINT BIOTECHNOLOGY LABORATORY, KATROUKHA, ALEXEI, KORPELA, TIMO, SEVERIN, EUGENII, SEVERIN, SERGEY
Application year: 1997
Main classification: G01N33
IPC codes: G01N33