In clinical medicine, the distribution of creatine kinase (CK) -MB, -MM or-BB isoenzyme activity in the circulation is used as an important and reliable parameter in the diagnosis of (acute) myocardial and brain infarction, acute or chronic inflammatory and non-inflammatory myopathies, brain and/or muscle traumas and, more recently, presence of lung, brain or prostate tumours. Since no uniform method is being applied for the discrimination between CK isoforms and the determination of their respective concentration the techniques of genetic engineering will be used to first produce biochemically pure CK-B. This material will then serve to prepare highly specific immunoprecipitating and immunoinhibiting monoclonal antibodies against CK-B. Thereafter it is planned to use these materials to set up an immunoenzymatic assay.
The project has started in 1990 and is expected to be completed within 1992.
The progress so far attained concerns: 1) the establishment of a procedure for routine production of beta-galactosidase CK-B fusion protein as CK-B antigenic material in E.Coli host bacteria; 2) the development of two alternative routes for the purification of this antigenic fusion protein, namely via HPLC (yielding protein with CK-B enzymatic activity) or via preparative SDS-acrylamide electrophoresis (yielding SDS-inactivated material).
The next steps will determine the procedure for upscaling the production and will evaluate the antigenic potential of the produced material. In parallel, a synthetic antigenic material was produced by conjugation of synthetic CK-B peptide to a Bovine Serum Albumin carrier molecule. This conjugate will be used in immunization experiments to produce highly specific antibodies to CK-B.