Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-10-30
1998-10-06
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 794, 436531, 436533, 436548, 53038826, G01N 33573
Patent
active
058174699
DESCRIPTION:
BRIEF SUMMARY
This application is a filing under 35 U.S.C. .sctn. 371 of PCT/EP 94/1337 filed on Apr. 28, 1994.
FIELD OF THE INVENTION
The invention concerns 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 a method for detecting CK-MB in a diagnostic test using these antibodies.
DESCRIPTION OF RELATED ART
Creatine kinase (ATP: creatine-N-phosphotransferase, EC 2.7.3.2) catalyzes the reversible phosphorylation of creatine in the presence of ATP to creatine phosphate. The enzyme is composed of two subunits of which an M (muscle-specific) and B (brain-specific) form is known which have a homology to one another of 78.7 %. The dimeric enzyme is composed either of two identical subunits (muscle-specific CK-MM isoenzyme and brain-specific CK-BB isoenzyme) or is present as a heterodimer. This heterodimeric CK-MB isoenzyme is specific for heart muscle and is detectable in serum above all in the case of injured myocardium e.g. cardiac infarction, a progressive muscular dystrophy or toxic myopathies. Due to its high specificity, the determination of CK-MB in serum is an important emergency parameter for diagnosis of a cardiac infarction. Therefore several methods for the determination of CK-MB in serum have already been described in which CK-MB is detected by an electrophoretic or ion-chromatographic separation, by a radioimmunoassay or an enzymatic determination of activity while inhibiting the M subunit with an antibody. These methods are, however, very time consuming. Since the determination of CK-MB is important particularly in emergency situations, a suitable test must not only be specific for CK-MB but capable of being carried out rapidly.
Immunoassays have therefore also been described in which CK-MB is detected by a sandwich assay using antibodies against CK-MM and CK-BB. Although theoretically only CK-MB should be detectable by this means, such a sandwich assay has proven to be very susceptible to interference by CK-MM and CK-BB since anti-CK-MM antibodies also cross-react with CK-BB and anti-CK-BB antibodies also cross-react with CK-MM. Therefore such a sandwich assay easily leads to false-positive results. Specific monoclonal antibodies for CK-MB were also developed by Vaidya et al. (Clin. Chem. 32, 1986, 657-663). One of these antibodies was characterized particularly well. This antibody is denoted the so-called "Conan" antibody and is considered to be a reference antibody for monoclonal antibodies against CK-MB. However, this and also a series of other monoclonal antibodies against CK-MB which have been developed in the meantime only have a low affinity to CK-MB and moreover all recognize the same epitope, the so-called "Conan" epitope of CK-MB, so that no sandwich immunoassay is possible with these antibodies.
In the previously described sandwich immunoassays only one CK-MB-specific monoclonal antibody is used. The second antibody is an antibody against the M or B subunit and thus also recognizes CK-MM or CK-BB.
SUMMARY OF THE INVENTION
The object of the invention was therefore to provide monoclonal antibodies against the CK-MB isoenzyme which do not bind to the CK-MM and CK-BB isoenzymes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This object is achieved by a monoclonal antibody which binds to the CK-MB isoenzyme but not to the B or M subunit alone of CK-MB or substantially to the CK-MM or CK-BB isoenzymes, the binding to CK-MB taking place in an equivalent manner to the binding of the monoclonal antibodies DSM ACC 2058, DSM ACC 2060 or DSM ACC 2168. This monoclonal antibody is obtainable by immunizing A/J mice (H-2.sup.a haplotype) with the CK-MB isoenzyme using aluminium hydroxide as an adjuvant, immortalizing the spleen cells of the immunized mice and cloning those immortalized cells which produce an antibody which binds to CK-MB but not to CK-MM or CK-BB and which is not displaced from the binding to CK-MB by the "Conan MB antibody" (ATCC HB 8939).
Such an antibody which reacts specifically with CK-MB but n
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Essig Ulrich
Hubner-Parajsz Christa
Lang Fridl
Vogel Rudolf
Boehringer Mannheim GmbH
Spiegel Carol A.
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