Abstract: Objective Construct heterodimeric CK-MB recombinant protein and prepare its specific geneengineered antibody as the diagnostic raw material of CK-MB quantitative detection kit, so as to assist theearly diagnosis of acute myocardial infarction in clinic, and to improve the accuracy of diagnosis. MethodMouse plasma cells were obtained by obtaining heterodimeric CK-MB through co-expression vector andanimal immunization, further, single mouse B cells specific for antigen were isolated using single-cellphotoconductive system, and genetically engineered antibodies were constructed after obtaining the genesequences of the variable regions of the light and heavy chains of the antibodies through single-cell PCR.Candidate antibodies were screened based on preliminary functional validation, and the optimal pairingscheme was further explored by checkerboard titration, and a CK-MB quantitative detection kit wasinitially assembled using the principle of double-antibody sandwich ELISA. Result CK-MB immunogenwas successfully obtained; single plasma cells specific for CK-MB antigen were screened by single B-cellphotoconductivity and two strains of mouse-derived genetically engineered antibodies were prepared, andboth of them had the ability of recognizing the natural CK-MB; two pairwise protocols were screened by double-antibody sandwich ELISA, and the R2of the fitted curves were both greater than 0. 99.Conclusion Mouse-derived genetically engineered antibody targeting CK-MB was successfully prepared,and the specificity and affinity of the antibody was preliminarily verified by ELISA, laying a solidfoundation for the further development of a new type of CK-MB quantitative detection kit with highspecificity and high sensitivity.

Key words: creatine kinase isoenzyme CK-MB, acute myocardial infarction, geneticallyengineered antibody

摘要： 目的 构建异源二聚体 CK-MB 重组蛋白并制备其特异性基因工程抗体,以此作为 CK-MB 定量检测试剂盒的诊断原料,从而辅助临床急性心肌梗死的早期诊断,提高诊断的准确性。 方法 通过共表达载体获取异源二聚体 CK-MB 并进行动物免疫,从而获取小鼠浆细胞;进一步使用单细胞光导系统分离抗原特异性小鼠单个 B 细胞,并通过单细胞 PCR 获取抗体轻重链的可变区基因序列后构建基因工程抗体。 基于初步的功能验证筛选出候选抗体,并进一步通过棋盘滴定法探究最佳的配对方案,并利用双抗夹心 ELISA 原理初步组装 CK-MB 定量检测试剂盒。 结果 成功获取 CK-MB 免疫原;通过单 B 细胞光导技术筛选出 CK-MB 抗原特异性小鼠单个浆细胞并制备出2 株小鼠源基因工程抗体,且均具有识别天然 CK-MB 的能力;经由双抗夹心 ELISA 法筛选出两种配对方案,其拟合曲线的 R2 均大于 0. 99。 结论 成功制备出靶向 CK-MB 的小鼠源基因工程抗体,经由 ELISA 初步验证抗体的特异性和亲和力,为进一步开发高特异性、高灵敏度的 CK-MB 新型定量检测试剂盒奠定了坚实基础。

关键词: 肌酸激酶同工酶 CK-MB, 急性心肌梗死, 基因工程抗体