Monoclonal antibodies against ck-mb

ES 2 189 802 T3

DESCRIPTION

Monoclonal antibodies against CK-MB.

The invention relates to monoclonal antibodies, which bind to the CK-MB isoenzyme, but not to the B or M subunit of CK-MB or to the CK-MM and CK-BB isoenzymes, as well as to a procedure for the detection of CK-MB in a diagnostic assay using these antibodies.

Creatine kinase (ATP: creatine-N-phosphotransferase, EC 2.7.3.2) catalyzes the reversible phosphorylation of creatine in the presence of ATP to form creatine phosphate. The enzyme consists of two subunits, of which an M form (specific for muscles) and a B form (specific for the brain) are known, which show a homology of 78.7% between them. The doimeric enzyme consists of either two idiosyncratic subunits (the muscle-specific CK-MM isoenzyme and the brain-specific CK-BB isoenzyme) or occurs as a heterodimer. This heterodimeric isoenzyme CK-MB is specific for cardiac muscle (myocardium) and is especially detectable in serum in the case of damage or damage to the cardiac muscle such as, for example, a cardiac infarction, a progressive muscular dystrophy or myopathies due to toxic causes. Due to its high specificity, the determination of CK-MB in serum is an important emergency parameter for the diagnosis of cardiac infarction. Therefore, some procedures for the determination of CK-MB in serum have already been described, in which CK-MB is detected through a separation by electrophoresis or by ion chromatography, through an immunological radiological assay. or an enzymatic determination of the activity, mediating inhibition of the M subunit, with an antibody. These procedures are however very long and tedious. Since the determination of CK-MB is particularly important in emergency situations, an appropriate assay must be able to be carried out not only in a specific way for CK-MB but also rapidly.

Therefore, they were also described in immunological assays, in which CK-MB is detected through a sandwich assay with antibodies against CK-MM and CK-BB. Even when, theoretically, it should be possible to detect only CK-MB, a sandwich test of this type has shown itself to be highly susceptible to disturbances against CK-MM and CK-BB, since antibodies against CK- MM cross-react also with CK-BB and antibodies against CK-BB also cross-react with CK-MM. Therefore, such a sandwich test leads to falsely positive results. Monoclonal antibodies were developed by Vaidya et al. (Clin. Chem. 32, 1986, 657-663) which are also specific for CK-MB. Of these antibodies, one of them was particularly well characterized. This antibody is designated as the so-called "Conan" antibody and is considered a reference antibody for monoclonal antibodies against CK-MB. This antibody, as well as a number of other monoclonal antibodies against CK-MB that have been developed in the meantime, however have only a low affinity for CK-MB and, furthermore, they all recognize the same epotope, the so-called "Conan" epotope. of CK-MB, so no immunological sandwich assay is possible with these antibodies.

In the case of the immunological sandwich assays described so far, only a monoclonal antibody specific for CK-MB is used. The second antibody is an antibody against the M or B subunit and therefore also recognizes CK-MM or CK-BB respectively.

It was therefore the mission of the invention to make available monoclonal antibodies against the CK-MB isozyme, which do not bind to the CK-MM and CK-BB isoenzymes.

The problem posed by this mission is solved by a monoclonal antibody, which binds to the CK-MB isoenzyme, but not to the B or M subunit of CK-MB alone or essentially does not bind to the CK-MM isoenzymes and CK-BB, fixing to CK-MB in a manner equivalent to fixing the monoclonal antibodies DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168. This monoclonal antibody is obtainable by immunization of A / J mice (from haplotype H-2^to) with the CK-MB isoenzyme by using aluminum hydroxide as an adjuvant, by immortalization of spleen cells (splenocytes) of immunized mice, and by cloning of those immortalized cells that produce an antibody that binds to CK-MB but does not to CK-MM or CK-BB and that it is not displaced by the "Conan MB antibody" (ATCC HB 8939) from the fixation to CK-MB.

Such an antibody, which reacts specifically with CK-MB but essentially not with CK-MM or CK-BB, must recognize a conformational epotope, which is formed by both the M subunit and the B subunit. Since the binding region of an antibody recognizes about 6 to 10 amino acids of the antigen as epotope, the two subunits of CKMB contribute only about 5 amino acids to this conformational epotope in each case.

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Furthermore, because of the stereo arrangement of the amino acid side chains and their hydrophilicity, only certain segments of a protein sequence act immunogenically. Thus, dimer antigens generally have only a very small number of conformational epatopes, the specificity of which the two subunits contribute in common. It was therefore surprising that monoclonal antibodies could be found against the CK-MB isoenzyme, which recognize four different conformation epitopes on the CK-MB isozyme, and are therefore suitable for a sandwich analysis or even for a formation of triplets. These four epitopes are characterized by the binding of the "Conan MB antibody" ATCC HB 8939 as well as the monoclonal antibodies according to the invention DSM ACC 2059 and / or DSM ACC 2057 for the first epitope, by the binding of the antibody DSM ACC 2058 for the second epitope, by the fixation of the antibody DSM ACC 2060 for the third epitope and by the fixation of the antibody DSM ACC 2168 for the fourth epitope.

It has been surprisingly shown that the antibodies DSM ACC 2058, DSM ACC 2060, DSM ACC 2057, DSM ACC 2059 and DSM ACC 2168 have a higher constant of association for CK-MB binding than monoclonal antibodies against the CK-MB, hitherto known. These monoclonal antibodies therefore react very rapidly with CK-MB and are therefore especially suitable for rapid diagnostic assay systems.

Therefore, a monoclonal antibody according to the invention against the isoenzyme CK-MB is preferred, which has an association constant for CK-MB fixation of at least 1x10⁵ mole^-1 xl xs^-1[s = seconds].

Preferably, the monoclonal antibodies according to the invention are of the IgGI isotype.

The monoclonal antibodies against CK-M according to the invention can be arbitrarily combined both with each other and also with known antibodies against CK-MB, for an immunologous sandwich assay for the detection of CK-MB, provided that that two antibodies are used, which recognize a different epitope on CK-MB. Of course, the antibodies according to the invention can also be combined with antibodies against the M or B subunit respectively of creatine kinase.

For an assay system that is as sensitive as possible, the antibodies should also have a high affinity for CK-MB. It has now been surprisingly shown that the aforementioned DSM ACC 2059 and DSM ACC 2057 monoclonal antibodies have a higher affinity constant than the hitherto known antibodies against this CK-MB epitope.

A further object of the invention is therefore a monoclonal antibody that binds to the CK-MB isoenzyme but not to the B or M subunit of CK-MB alone or does not essentially bind to the CK-MM and CK-isoenzymes. BB, the fixation to CK-MB being carried out in a manner equivalent to the fixation of the monoclonal antibody DSM ACC 2057 or DSM ACC 2059, which presents an affinity constant of at least 1 x 10⁸ mole^-1 xl for the CK-MB. This monoclonal antibody is obtainable by immunization of A / J mice (of the H-2 haplotype^to) with the CK-MB isoenzyme using aluminum hydroxide as adjuvant, by immortalization of spleen cells of immunized mice and by cloning of immortalized cells that produce an antibody, which binds to CK-MB but essentially not to CK-MM or CK-MB, which is displaced from CK-MB binding by the "Conan MB antibody" (ATCC HB 8939) and exhibits an affinity constant of at least 1 x 10⁸ mole^-1xlparalaCK-MB. The determination of the affinity constant was carried out in this case through the BIAcore® system (from Pharmacia LKB) using the Kinetic Evaluation Kit Software (from Pharmacia LKB, identification number BR- 1000-19).

The monoclonal antibodies DSM ACC 2057 and DSM ACC 2059, mentioned above, also have a dissociation constant of less than 4 x 10^-4 s^-1. This means that the complex of an antibody and CK-MB dissociates again only very slowly. This is important for certain immunoassay procedures, such as eg. the immunological enzyme maetric assay IEMA (from Immuno Enzyme Metric Assay), since a rapid dissociation of the complex between the antibody and the CK-MB will lead to a competitive reaction between the binding of the labeled antibody to the analyte or to the immobilized antigen and therefore to a falsification of the value of the measure.

A preferred object of the invention is therefore a monoclonal antibody according to the invention against the isozyme CK-MB, which binds to CK-MB in a manner equivalent to that of the antibody.

ES 2 189 802 T3 monoclonal DSM ACC 2057 or DSM ACC 2059 and exhibiting a dissociation constant of less than 4 x 10^-4 s^-1. The monoclonal antibodies according to the invention are particularly preferred, which furthermore have an association constant for binding to CK-MB of at least 1 x 10⁵ mole^-1xlxs^-1 .

The antibodies according to the invention can be obtained by immunization of A / J mice (haplotype H-2^to) with the isozyme CK-MB. It has surprisingly been found that in the case of immunization of this breed of mouse an essentially higher antibody titer is obtained than with the Balb / c mice which are usually used for this. Furthermore, it has proven to be particularly advantageous for the production of the antibodies according to the invention against a conformational epotope to adsorb CK-MB for immunization to aluminum hydroxide and Bordetella pertussis. After approximately four months, the spleen cells of the immunized mice are immortalized and those immortalized cells that produce the desired antibodies are cloned. The immortalization of the spleen cells was carried out according to procedures known to a person skilled in the art; preferably the coells are fused with myeloma cells. Those immortalized cells, which produce the desired antibodies, are identified by parallel determination of the reactivity of a sample of the culture supernatant respectively with an immobilized CK-MB, CK-MM or CK-BB. The desired antibodies against a conformational epotope of CK-MB react with CK-MB, but essentially not with CK-MM or CK-BB. The designation of "essentially no" means that the reaction with CK-MM and CK-BB was so low that in an immunological test system for the determination of CK-MB the disturbances caused by the presence of CK -MM and CK-BB in their physiologically present concentration amount to less than 1%, preferably less than 0.1%. The reaction with CK-MM should be minimally small in such a case, since CK-MM occurs in high concentrations in serum or plasma. Since CK-BB occurs in serum or plasma only in very small concentrations, a small reaction of antibodies with this isoenzyme is not relevant. The corresponding immortalized cells are cloned according to usual procedures, eg. by isolation through a fluorescence activated cell sorter. For the identification of those clones that produce an antibody that binds to CK-MB in a manner equivalent to the monoclonal antibody DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168, a competitive assay system is used. . To do this, for example with the help of an enzyme-linked immunosorbent assay, it is verified to what extent the antibody competes with the "Conan antibody MB" (ATCC HB 8939) for binding to CK-MB. CK-MB is then incubated with the "antibody Conan MB ”(ATCC HB 8939) in labeled form and in excess of the antibody taken into consideration. By immobilization of the complexes formed, by separation of the solid phase from the liquid phase and by detection of the label attached to one of the two phases, it can be easily verified whether the antibody taken in consideration has been displaced from the fixation. by "Conan MB antibody" (ATCC HB 8939). If this is not the case, then a binding to CK-MB is present that corresponds with a high probability equivalent to binding to the monoclonal antibody DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168.

For the identification of clones, which produce an antibody that has the desired association constant or dissociation constant of the binding to CK-MB or the desired affinity for CK-MB, then in a sample of the culture supernatant the association constant, dissociation constant and respectively the affinity of the antibody formed for CK-MB. This determination is preferably carried out using the BIAcore® system (Pharmacia LKB, Kinetic Evaluation Kit, Identification No. BR-1000-19). In this way, the hybridoma cell lines DSM ACC 2059, DSM ACC 2057, DSM ACC 2058, DSM ACC 2060 and DSM ACC 2168 could be obtained.

A particularly preferred object of the invention is constituted by monoclonal antibodies, which are obtainable from one of the hybridoma lineages DSM ACC 2059, DSM ACC 2057, DSM ACC 2058, DSM ACC 2060 and / or DSM ACC 2168, as well as the mentioned lineages hybridoma.

A further object of the invention is a process for the production of monoclonal antibodies, which bind to the CK-MB isoenzyme, but not to the B or M subunit of CK-MB alone or do not essentially bind to the CK-isoenzymes. MM and CK-BB, carrying out this fixation to CK-MB in a manner equivalent to the fixation of the monoclonal antibodies DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168 by immunization of A / J mice (haplotype H -two^to) with the isoenzyme CK-MB using aluminum hydroxide as adjuvant, by immortalization of spleen cells of immunized mice, by cloning of those immortalized cells that produce an antibody, which binds to CK-MB, but not binds essentially to CK-MM and CK-BB and is not displaced from CK-MB binding by the "Conan MB antibody" (ATCC HB 8939), and by isolation of the monoclonal antibody from the cloned cells or their culture supernatant, according to

ES 2 189 802 T3 with known procedures.

In a special embodiment of the method according to the invention, clones producing an antibody are selected which have an association constant for binding to CK-MB of at least 1 x 10⁵ mole^-1 xlxs^-1 or a dissociation constant of less than 4 x 10^-4 s^-1 or an affinity constant of at least 1 x 10⁸ mole^-1 xl.

With the aid of the monoclonal antibodies according to the invention, immunological test procedures for the diagnostic determination of CK-MB are now also possible, which require two CK-MB-specific antibodies with high affinity to different epitopes.

A further object of the invention is therefore the use of a monoclonal antibody according to the invention for the determination of CK-MB in a diagnostic test.

This determination can be carried out by means of all common immunological tests, such as, for example, ELISA, fluorescence immunological test, electrochemiluminescence immunological test (international patent applications WO 86/02734, WO 87/06706, WO 92/14138), radiological immunological assay, immunological fluorescence polarization assay, CEDIA (Henderson et al., Clin. Chem. 32 (1986), 1,637-1641 and US patent document US 4,708,929), IEMA or EMIT. The test can be carried out both in the form of a homogeneous test system, eg by means of a competitive immunological test, and also in the form of a heterogeneous test system. Preferably, the test was carried out with immobilization of one of the participants in the reaction. A determination by means of the so-called LPIA test system (Latex Particle Immunoassay = latex particle immunoassay) is particularly preferred. For this, two antibodies against different epotopes of CK-MB are first bound to a universal latex matrix with streptavidin. The determination of CK-MB was then carried out by incubating these conjugates with the sample or with a CK-MB standard and measuring the increase in extinction, which has been produced by agglutination.

A further object of the invention is therefore a method for the diagnostic detection of CK-MB in a homogeneous diagnostic test system, in which at least one antibody according to the invention binds to a loatex particle and subsequently incubation with the sample solution as well as with an additional latex-fixed antibody, which binds to CK-MB, without displacing the first antibody from its binding to CK-MB, The agglutination of the antibodies fixed to loatex is determined, as a measure of the concentration of CK-MB in the sample.

In this case, a serum sample is usually used as the sample solution. As a second antibody bound to latex particles, any antibody that reacts with CK-MB can be used in principle, that is to say also an antibody against the B or M subunit of creatine kinase. Preferably, however, a very similar antibody according to the invention against a conformational epotope of CK-MB is also used as a latex-bound second antibody. Especially preferably, as one of the two antibodies bound to loatex, an antibody is used which is produced by the hybridoma cell line DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168. The antibodies can be used in this case both in in the form of whole antibodies as well as in the form of functional antibody fragments. Fab 'fragments are preferably used.

A further subject of the invention is a method for the detection of CK-MB in a homogeneous diagnostic test system according to the CEDIA method, in which an antibody according to the invention is used. In the CEDIA procedure, certain enzymes such as β-galactosidase are used for the detection, which are present in two constituents in each case enzymatically inactive, a large polypeptide (acceptor of enzymes EA) and a small polypeptide (donor of enzymes ED). These constituents spontaneously associate with the enzymatically active proteon. A peptide antigen is attached to ED, in the case of CK-MB, this isozyme or a fragment of it, which contains the specific epotopes for CK-MB, in such a way that the association of an ED with an AD is not prevented. . This association is inhibited in the event that an antibody against the peptide antigen binds to the complex of peptide antigen and an ED. In a reagent solution in which the EA, the peptide antigen complex and the ED are presented and an antibody specific for the peptide antigen, no active enzyme can be formed. After adding the sample solution, which contains peptide antigens, or in the case of the detection of CK-MB the isoenzyme CK-MB, CK-MB competes with the peptide antigen complex and ED for fixation. to the antibodies according to the invention, whereby the active complex of an ED and an EA can be formed. The measurement signal is therefore proportional to the amount of the CK-MB isoenzyme present in the sample. In the case of the procedure

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CEDIA is a suitable antibody, which binds to CK-MB in a manner equivalent to that of the monoclonal antibody DSM ACC 2057 or DSM ACC 2059, and has an affinity constant for CK-MB of at least 1 x 10⁸ mole^-1 x l. Especially preferred is the monoclonal antibody DSM ACC 2059, which has an especially high affinity constant of 7.0 x 10⁸ mole^-1 xl.

A further object of the invention is a method for the diagnostic detection of CK-MB by means of a sandwich assay, in which at least one antibody according to the invention is bound to a solid phase, incubated with the sample solution as well as with an additional labeled antibody, which binds to CK-MB, without displacing the first antibody from its binding to CK-MB, and after having separated the solid phase and the liquid phase, The labeling in one of the two phases is determined as a measure of the concentration of CK-MB in the sample. Preferably, in this case, an antibody is used as one of the two antibodies, which is produced by the hybridoma line DSM ACC 2058, DSM ACC 2060 or or DSM ACC 2168.

The immobilization of the first antibody to the solid phase was carried out in a manner known to a person skilled in the art. Preferably, the antibody is biotinylated and fixed to a streptavidin-coated solid phase. The labeling of the second antibody was carried out in the usual way, eg. by direct labeling with an enzyme, a fluorescent or chemiluminescent dye, or by fixing an additional antibody, which was directed against the second antibody and which had been labeled in a corresponding way.

Detection by means of the immunological electro-chemiluminescence (ECL) assay, as described in WO 85/02734, WO 87/06706 or WO 92/14138, is further preferred. For this, an antibody against CK-MB is first bound to a magnetic particle. Preferably, fixation was via streptavidin and biotin. Magnetic particles coated with streptavidin and a biotinylated antibody against CK-MB are used. The second antibody according to the invention against CK-MB must recognize a different epitope than the first antibody. This second antibody is coupled with the label. As labeling an electro-chemiluminescent compound is used, as described in WO 86/2734 and WO 87/06706. Preferably, tris- (bispyridyl) ruthenium is used as the label. The coupling of the label to the antibody was carried out according to state-of-the-art methods. The two antibodies according to the invention can be incubated with the sample and the magnetic particles at the same time or consecutively. An apparatus is used for the measurement, as described in WO 90/11511.

Preferably, in the case of the ECL immunoassay, a first antibody is used, which binds to CK-MB in a manner equivalent to DSM ACC 2069 or DSM ACC 2057 monoclonal antibodies. The second antibody binds to CK-MB. a distinct and preferred epitope in a manner equivalent to that of the monoclonal antibody DSM ACC 2058.

In the case of the immunological sandwich assay, the use of two monoclonal antibodies has proven to be especially advantageous, both of which bind to the CK-MB isoenzyme, but not to the B or M subunit alone of the CK-isoenzyme. MB and do not bind essentially to the isoenzymes CK-MM and CK-BB and do not compete with each other for the same binding to CK-MB, and of which at least one has an affinity constant of at least 1 x 10⁸ mole^-1 xl for CK-MB. In a particularly preferred manner, at least one of these antibodies has an association constant of 1.9 x 10⁵ mole^-1 xlxs^-1.

The hybridoma cell lines according to the invention, DSM ACC 2059, DSM ACC 2057, DSM ACC 2058 and DSM ACC 2060 mentioned above, were deposited on 02/03/1993, and the hybridoma cell line DSM ACC 2168 was deposited on 04/19. 1994 in the German Collection for Cell Cultures and Microorganisms SA [Deutsche Sammlung fur Zellkulturen und Mikroorganismen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig].

The invention is explained in more detail by the following Examples.

Example 1

Production of monoclonal antibodies against the isozyme CK-MB

a) Immunization of mice

A / J mice aged 12 weeks are first immunized intraperitoneally with

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100 μg of CK-MB (from Aalto, Dublin). After 6 weeks, two additional immunizations follow intraperitoneally at one-month intervals. In this case, each mouse is administered 100 μg of CK-MB adsorbed to aluminum hydroxide (from Paesel, Frankfurt) as well as 10⁹Bordetella pertussis germs (from Behring, Frankfurt). The last two immunizations were then carried out intravenously each time with 100 µg of CK-MB in a PBS buffer on days 3 and 2 before fusion.

b) Fusion and cloning

The fusion of spleen cells from mice immunized following 1. with myeloma cells was carried out using the method of Galfríe, Methods in Enzymology 73, 1981, 3. In this case, approximately 1 * 10⁸ spleen cells of immunized rats are mixed with 2 * 10⁷ myeloma cells (P3X63-Ag8-653, ATCC CRL 1580) and separated by centrifugation (for 10 min at 300 xg and 4^°C). The cells are then washed once with RPMI-1640 medium without fetal calf serum (FKS, from Fotales KalberSerum), and again separated by centrifugation at 400 xg in a sharp tube with a capacity of 50 ml. The supernatant is turned over, the cell pellet is slightly loosen by tapping, 1 ml of PEG (4,000 molecular weight, Merck, Darmstadt) is added thereto and mixed by pipetting. After 1 min in a water bath at 37 ° C, 5 ml of RPMI 1640 without FKS are added dropwise at room temperature over a period of 4-5 min. After that, 5 ml of RPMI 1640 with 10% FKS are added dropwise in about 1 min, mixed thoroughly, made up to 50 ml with medium (RPMI 1640 + 10% FKS) and then centrifuged for 10 min at 400 xg and 4^°C. The sedimented cells are collected in RPMI 1640 medium with 10% FKS and seeded in hypoxanthine-azaserine selection medium (100 mmol / l hypoxanthine, 1 μg azaserine in RPMI 1640 + 10% FKS) . Interleukin 6 (Boehringer Mannheim GmbH, Catalog No. 1271172, 100 U / ml) is added to the medium as growth factor.

After approximately 10 days, the primary cultures are tested for specific antibody synthesis and cross-reaction (see Example 2). CK-MB positive primary cultures, which do not show any essential cross-reactions with the CK-MM and CK-BB isoenzymes, are cloned into 96-well cell culture plates by means of a fluorescence activated cell sorter. In this case, interleukin-6 is added to the medium as a growth additive (Boehringer Mannheim GmbH, N^° catylogo 1271172, 100 U / ml). Of those clones that produce an antibody against a conformation epitope of CK-MB (determination according to Example 2), those cultures are selected that present an antibody with an affinity of at least 9 x 10^-7 mole^-1 xl for CK-MB (determination according to Example 3) and / or have an antibody, which recognizes an epitope other than the "Conan MB antibody" (determination according to Example 4).

c) Obtaining immunoglobulin from cell culture supernatants

The hybridoma cells obtained are seeded with a density of 1 x 10⁵ cells per ml in RPMI 1640 medium with 10% FKS and reproduced for 7 days in a fermenter (from Thermodux, Wertheim, Main, model MCS-104XL, N^° custom 144-050). Concentrations averaged 100 μg of monoclonal antibody per ml are reached in the culture supernatant. The purification of this antibody from the culture supernatant was carried out according to usual methods in protein chemistry (eg according to Methods in Enzymology 121 (1986), 587-695).

Example 2

Determination of the specificity of the antibodies produced

In order to determine the specificity of the antibodies in the culture supernatant of the hybridoma cells, the reactivity with CKMB, CK-MM and CK-BB is determined in three parallel ELISA runs. To do this, 96-well microtiter plates (Nunc) are first coated with 200 μl / well of thermo-RSA-streptavidin (10 μg / ml of a coating buffer = 0.2 mol / l of carbonate or bicarbonate of sodium, pH 9.3 - 9.5, Boehringer Mannheim GmbH, N^° 0725559) (incubation for 1 h at room temperature with shaking) and washed 1 time with a mixture of 0.9% NaCl and 0.05% Tween 20. In parallel, the CK-MB, CK- MM and CK-BB (from Aalto, Dublin, N^° catalog numbers 10901, 10217 and 10803) with the Nhydroxy-succinimide ester of D-biotinyl-e-amido-caproic acid (Boehringer Mannheim GmbH, Catalog No. 01008960) according to the manufacturer's data. Biotinylated creatine kinase isoenzymes are collected, at a concentration of 250 ng / ml of PBS, with a mixture of 0.05% Tween 20 and 1% alumina of

ES 2 189 802 T3 bovine serum (RSA), in each case at a rate of 100 µΐ / well of the microtiter plates coated with thermo-RSA-streptavidin and incubated with shaking for 1 h at room temperature. It is then washed 3 times with a mixture of 0.9% sodium chloride and 0.05% Tween 20. In each case 100 μΐ of the antibody solution to be investigated is added in parallel batches in each case to a well coated with CK-MB, CK-MM or CK-BB respectively, and incubated with shaking at room temperature. for 1 h. After having washed 3 times with a mixture of 0.9% sodium chloride and 0.05% Tween 20, for the detection of the antibody bound from the sample, 100 μ are added in each case! of a POD-labeled Fab fragment of a polyclonal antibody from a lamb against F_CTof mouse (Boehringer Mannheim GmbH, ID No. 1047523 corresponding to 100 mU), incubated with shaking at room temperature for 1 h and then washed 3 times with a mixture of 0.9% sodium chloride and 0.05 % Tween 20. Finally, 100 μl / well of ABTS® (Boehringer Mannheim GmbH, N^° catalog numbers 1204521 and 1204530) and after 30 min the extinction at 450/490 nm is measured at room temperature in a MR700 Microplate Reader from Dynatech.

Example 3

Determination of the affinity, the association and dissociation constants as well as the accessibility to a sandwich of the antibodies produced

The determination of the affinity, the association and dissociation constants, as well as the accessibility to a sandwich of the antibodies produced was carried out according to the BIAcoreC system of the Pharmacia LKB entity. In this case, the CK-MB isoenzyme is covalently attached to the surface layer of a so-called sensor chip and thereby immobilized. In the case of transferring a solution of the antibody to be determined, the antibody being fixed by non-covalent interaction forces to the immobilized CK-MB, the mass density in the surface layer is increased. The increase in mass density can be directly monitored by "Surface Plasmon Resonance". Depending on the number of binding sites on the immobilized reaction partner, third and fourth reaction partners can also be deposited. Its fixation can be observed in the same way as that of the first participant in the reaction in terms of kinetics and quantity.

All the participants in the reaction, with the exception of the covalently fixed CK-MB, can be detached again without damage from the CK-MB with simple means, so that together with the same primary phase other experiments can be carried out. additional fixation in ideal marginal conditions.

For the fixation of the CK-MB to the surface layer of the sensor chip, a solution of 20 μg / ml of CK-MB (from Aalto, lot no. 20410) in a mixture of 10 mmol is passed through the sensor chip. / l of HEPES, 3.4 mmol / l of EDTA and 150 mmol / l of NaCl, pH 7.4, with a flow rate of 2 μl / min.

The following antibodies are then added and the association and dissociation constants are determined respectively for the binding to CK-MB as well as the affinity of the antibodies for CKMB by means of the BIAcore® system according to the manufacturer's data (Pharmacia LKB, Software Kinetic Evaluation Kit, N^° identification BR-1000-19). The as-determined values ​​of the antibodies according to the invention, as well as of the "Conan MB antibody" ATCC HB 8939 and of a commercially available antibody against CK-MB (BP 172-A2720 from Bios Pacific) are compiled in the following Table 1 .

ES 2 189 802 T3

TABLE 1

<td>Antibody</td><td>Constant of association (mole^-1 xlxs^-1)</td><td>Dissociation constant (s^-1)</td><td>Affinity constant (mol^-1 xl)</td>

<td>DSM ACC 2059</td><td>1.9 x 10⁵</td><td>2.7 x 10^-4</td><td>7.0 x 10⁸</td>

<td>DSM ACC 2057</td><td>4.4 x 10⁴</td><td>1.8 x 10^-4</td><td>2.4 x 10⁸</td>

<td>DSM ACC 2058</td><td>1.8 x 10⁵</td><td>3.5 x 10^-3</td><td>5.0 x 10⁷</td>

<td>DSM ACC 2060</td><td>6.4 x 10⁴</td><td>2.9 x 10^-3</td><td>2.2 x 10⁷</td>

<td>ATCC HB 8939</td><td>3.4 x 10⁴</td><td>4.1 x 10^-4</td><td>8.2 x 10⁷</td>

<td>BP173-A2720 (Bios Pacific)</td><td>4.1 x 10⁴</td><td>1.7 x 10^-3</td><td>2.5 x 10⁷</td>

In a similar way, the accessibility of the antibodies according to the invention to a sandwich can be determined by means of the BIAcore® system. To do this, first a polyclonal antibody against murine IgGI or a polyclonal antibody against murine Fc-τ is immobilized on the sensor chip. It is then first loaded with a first antibody against CK-MB to be investigated, up to a quantity of 1,000 resonance units and the still free uptake antibody is blocked by transfer of a non-cross-reactive immunoglobulin. with CK-MB. After transfer of a solution containing the CK-MB, the association is monitored until equilibrium is reached, then the potential sandwich partners to be investigated are added and the association of the sandwich complex is monitored until the Balance. In this case, it has been found that the combinations of the antibodies according to the invention, which are listed in Table 2 below, are suitable for detecting CK-MB. The combinations of DSM ACC 2057 with DSM ACC 2058 or DSM ACC 2060 as well as the combinations of DSM ACC 2059 with DSM ACC 2058 or DSM ACC 2060 respectively have proven to be particularly suitable. On the contrary, with the current standard antibody against CK-MB (ATCC HB 8939), only a weak formation of a sandwich could be observed, which should not be considered as sufficient for a detection as a reliable diagnosis.

TABLE 2

<td>First antibody</td><td>Second possible antibody for a sandwich</td>

<td>DSM ACC 2057</td><td>DSM ACC 2058 DSM ACC 2060</td>

<td>DSM ACC 2059</td><td>DSM ACC 2058 DSM ACC 2060</td>

<td>DSM ACC 2059</td><td>DSM ACC 2057</td>

Using the monoclonal antibodies DSM ACC 2057 and DSM ACC 2059, a triplet formation can also be achieved with the combinations indicated in Table 3 below, in which three antibodies bind to CK-MB. In this way, a further increase in sensitivity and specificity can be achieved in comparison with the normal sandwich test.

TABLE 3

<td>First antibody</td><td>Second antibody</td><td>Third antibody</td>

<td>DSM ACC 2057 DSM ACC 2059</td><td>DSM ACC 2058 DSM ACC 2058</td><td>DSM ACC 2060 DSM ACC 2060</td>

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Example 4

Determination of overlap of epitopes of antibodies against CK-MB

For the detection of the overlapping of epitopes of an antibody with an antibody according to the invention, a competitive enzyme-linked immunosorbent assay is carried out. To do this, CK-MB is first biotinylated with the N-hydroxy-succinimide ester of D-biotinyl-e-amido-caproic acid (Boehringer Mannheim GmbH, N.^° catalog number 1008960) according to the manufacturer's data. 300 ng of this biotinylated antigen are fixed in a volume of 100 μ! of PBS by incubation for 1 hour at room temperature, to a microtiter plate coated with streptavidin (produced according to EP-A 0,344,578). After having washed 3 times with a mixture of PBS and 0.05% Tween 20, it is incubated simultaneously at room temperature for 90 min with the reference antibody (eg DSM ACC 2059) that had been labeled with peroxidase. (final concentration 52 mU / ml), and with the antibody to be evaluated. After having washed again 3 times with a mixture of PBS and 0.05% Tween 20, it is incubated at room temperature for 30 min with the ABTS® enzyme substrate solution in a buffer containing sodium perborate, and then extinction at 405 nm is measured as a measure of the amount of bound, POD-labeled monoclonal antibody. This value is compared with the extinction, which is obtained in the case of an incubation with the labeled monoclonal antibody alone. When at least a 50% competition can be recognized, up to an excess of 10⁵ times the amount of antibody to be evaluated compared to the labeled antibody, there is an overlap of epitopes.

Antibodies from the cell lines DSM ACC 2057 and DSM ACC 2059 showed strong competition, that is, they recognize and respectively bind to the same epitope, which is also recognized by the so-called "Conan antibody". Antibodies from the DSMACC2058, DSM ACC 2060 and DSM ACC 2168 cell lines do not compete with the antibodies from the other two cell lines (DSM ACC 2057 and DSM ACC 2059). With the exception of the two antibodies from the DSM ACC 2060 and DSM ACC 2168 cell lines, these three antibodies do not show any competition. The antibodies DSM ACC 2060 and DSM ACC 2168 show little competition, which leads to the conclusion that the two epatopes only slightly overlap. Therefore, it is not advantageous to use these two antibodies for example in common in a sandwich assay. However, in combination with another antibody according to the invention, this antibody can be used very well.

Example 5

Determination of CK-MB through an LPIA test system

First, two antibodies according to the invention, suitable for a sandwich assay system (see Example 3), are chosen and Fab 'fragments are biotinylated according to the data presented in EP-A-0,464,554.

In each case 20 μ! of a CK-MB standard (0, 110 and 220 ng of CK-MB / ml of human serum) or the sample to be analyzed is incorporated by pipetting as a sample in a Hitachi 717 photometer at time t = 0. For this, 330 μ! Are added by pipetting immediately through the apparatus. from Reagent 1 (reaction buffer = 50 mmol / l Tris / HCl, pH 7.5, 75 mmol / l sodium chloride, 3% PEG 35,000, 1% Pluronic F68, 0.1% Brij 35 and 0.1% sodium azide). At time t = 4.5 min, 50 μ! from Reagent 2 (latex suspension); latex with streptavidin 0.083% previously coated in each case with 1.6 μg of antibodies / ml in 200 mmol / l of glycine, pH 7.5, 2% sucrose, 0.5% RSA I (RSA, step purity I, with 0.1% sodium azide). The increase in agglutination is then measured bichromatically at 340 nm (main wavelength) and at 700 nm (secondary wavelength) (measurement temperature 37^°C). As magnitude of the signal, the extinction difference between the moments t = 5.3 min and t =

9.5 min. In this case, the combination of the antibodies DSM ACC 2059 and DSM ACC 2058 (corresponding to a high dynamics (difference between the 2^° measurement value and vacuum value) of 342 mE) as well as a high initial rise of the calibration curve (2.3 mE x ml / ng).

ES 2 189 802 T3

Example 6

Determination of CK-MB through a sandwich assay system according to the electro-chemiluminescence procedure

The ECL procedure was carried out in a manner corresponding to WO 86/02734, WO 87/06707 and WO 92/14138. The monoclonal antibody DSM ACC 2059 was biotinylated according to state-of-the-art methods (Solution 2). The monoclonal antibody DSM ACC 2058 was routenylated with DSS (disuccinyl substrate) ((Tris) (2,2'-bipyridyl) -ruthenium-chloride-hexahydrate) (Solution 3). Both antibodies were incubated for 5 minutes with the sample containing CK-MB in Solution 1 and then streptavidin-coated magnetic particles (Dynabeads M-280 streptavidin from Deutsche Dynal GmbH, Germany) (Solution 4) were added and incubated renewed for 5 minutes.

The reaction is terminated by suction filtration of the reaction mixture into the measuring cell. For the application, an Origen 1.0 device from Igen, USA was used. In the measuring chamber, the magnetic particles are retained by means of a magnet on the electrode. The remaining reaction mixture is suction filtered and the measuring chamber is filled with assay buffer (Solution 5). The emission of light is excited by the application of an electrical voltage.

The following solutions were used: #

Solution 1

Incubation buffer:

100 mM sodium phosphate pH 7.0

0.1% RSA

0.1% methyl isothiazolone

0.1% sodium benzoate

Solution 2

Biotinylated MAK (monoclonal antibody) DSM ACC 2059 2 μg / ml in an incubation buffer Solution 3

Ruthenium-bispyridyl labeled MAK DSM ACC 2058 2 μg / ml in incubation buffer Solution 4

500 μg / ml of streptavidin-coated magnetic particles in:

mM Hepes, 0.1% RSA, 0.1% Thesit, 0.1% Chloroacetamide,

0.01% methyl isothiazolone, pH 7.0

Solution 5

Anaolysis buffer: pH 6.8

0.16 M tris-propyl-amine, 0.2 M dipotassium hydrogen phosphate,

Polidocanol (Thesit) 0.1%, Oxaban A 0.1%.