人心肌线粒体肌酸激酶的提纯及抗体制备

The purification of human sarcomeric mitochondrial creatine kinase and preparation of the specific antibody

目的 从人心肌中分离纯化肌小节型线粒体肌酸激酶 (sMtCK) ,制备抗血清 ,从抗血清中制备对sMtCK有特异免疫反应的抗体 ,建立酶联免疫吸附试验 (ELISA) ,测定血清sMtCK。方法 采用超低温高速离心、硫酸铵分级提取、SepharoseCL6B层析等技术分离纯化sMtCK ,免疫新西兰大白兔。应用自制抗sMtCK建立ELISA检测健康体检者、心肌炎及急性心肌梗死 (AMI)患者血清sMtCK。结果 经聚丙烯酰胺凝胶电泳(SDS PAGE)和Westernblot鉴定 ,纯化的sMtCK为单一蛋白质条带。制备的抗sMtCK多克隆抗体在琼脂糖免疫扩散实验和ELISA中的抗体效价分别为 1∶32和 1∶80 0。自制抗体建立的ELISA检测心肌炎和AMI患者的血清sMtCK阳性率分别为 5 1.6 % (16 / 31)和 5 4 .1% (2 0 / 37) ,与健康对照组相比差异有显著性 (P <0 .0 1)。结论 经离心、提取、纯化所得的sMtCK是免疫纯。建立的ELISA检测血清sMtCK有助于心肌炎和AMI的诊断。

Objective Purification of sarcomeric mitochondrial creatine kinase (sMtCK) from cardiac muscle and preparation of the specific antibody against sMtCK and establishment a method of ELISA to test serum sMtCK. Methods The sMtCK of human myocardium was isolated by ultra low temperature high speed centrifugation and precipitated by ammonium sulfate and then chromatographically purified with sepharose CL6B column and immunized New Zealand rabbit. A method of ELISA for testing sMtCK was developed to detect the serum sMtCK of nomal human and patient with myocarditis and AMI. Results With SDS PAGE and Western blot, the sMtCK was identified to have only one band. The Ouchterlony test and ELISA of prepared specific antibody against sMtCK showed that the titer was 1∶32 and 1∶800 respectively. The results given by ELISA established with prepared antibody showed that the positive rate of sMtCK of 31 patients with myocarditis and 37 with AMI were 51.6%(16/31) and 54.1%(20/37) respectively, and had a statistical significance compared with the control group ( P <0.01). Conclusions The purified sMtCK is immunologically pure. The method of ELISA established to test serum sMtCK is to clinically useful.