ENZYME IMMUNOASSAY SYSTEM FOR MEASUREMENT OF THREE FORMS (MM, MB AND BB) OF RAT CREATINE KINASE

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A highly sensitive sandwich-type enzyme immunoassay system for the assay of rat creatine kinase (CK) MM, MB and BB isozymes was developed by use of β-D-galactosidase from Escherichia coli as label and the purified antibodies raised in rabbits by injecting CK-MM or CK-BB. The antibodies specific to each subunit were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column ofrespective antigen (CK-MM or CK-BB)-coupled Sepharose. The CK-MM and CK-BB assays were performed with the respective antibody F(ab’)₂ fragments-immobilized polystyrene balls and the same antibody Fab’ fragments labeled with β-D-galactosidase from Escherichia coli. The CK-MB was assayed with the polystyrene balls with either antibody (anti-CK-M or anti-CKB) and another antibody (anti-CK-B and anti-CK-M, respectively) labeled with galactosidase. The minimum detection limit of each assay was 1 to 10 pg or (10 to 100 amol) per tube. The assays for CK-MM and CK-BB were cross-reacted about 12% with CK-MB, but the assay for CK-MB was specific showing no cross-reactivity with and no interference by CK-MM and CK-BB under the limited conditions. Therefore, the concentrations of CK-MM and CK-BB were able to estimate by subtracting the cross-reacting CK-MB in the samples. The concentrations of three forms of CK in various rat tissues were determined by the present assay system. High concentrations of CK-MM were observed in the heart and tissues composed of striated muscle or chondrocytes. CK-MB was also present at relatively high levels in the above-mentioned tissues, while CK-BB was mostly located in the central nervous system and tissues composed of smooth muscle. These results are in line with the previous reports on the distribution of CK isozymes in human tissues.