Preparation of Creatine Kinase Isoenzyme MB (CK-MB) Monoclonal Antibody and Establishment of Detection Method

In this paper by preparing monoclonal antibodies with high specificity, a double antibody sand-wich ELISA method for quantitative determination of CK-MB was preliminarily established in the present paper, which laid an important foundation for the domestication of CK-MB reagents and raw materials. Five randomly selected Balb/c female mice (6~8 weeks old) were immunized by using the purchased CK-MB as immunogen. By  limited dilution method, indirect and capture ELISA method, three cell lines which could secrete antibodies stably were screened, named as 2F6, 2H3 and 2H9, respectively. The monoclonal antibody subtypes secreted by these three cell lines were all IgG3, with ascites titers of  201׃1000,  15׃1200 and  201׃1000, respectively. The ascites of mice produced by three hybridoma cells was purified by affinity chromatography. The concentrations of monoclonal antibodies 2H3, 2F6 and 2H9 determined by spectrophotometer were 5 mg/mL, 6 mg/mL and 6 mg/mL, respec-tively. The results of SDS-PAGE showed that the ascites of monoclonal antibody was purified successfully and two bands of light chain and heavy chain could be clearly observed. Results of Western blot showed that monoclonal antibodies 2F6, 2H3 and 2H9 could specifically recognize CK-MB protein. Indirect and capture ELISA detection showed that monoclonal antibody 2H3 reacted only with CK-MB and CK-BB capture ELISA; monoclonal antibody 2H9 reacted only with CK-MB and CK-BB capture ELISA; and monoclonal antibody 2F6 reacted only with CK-MB and CK-MM capture ELISA. The stability test showed that all the three hybridoma cells could secrete anti-CK-MB monoclonal antibodies stably. The acquisition of monoclonal antibodies in high quality lays a foundation for the establishment of CK-MB detection method.