重组人心肌肌钙蛋白Ⅰ单克隆抗体的制备及鉴定

目的制备重组人心肌肌钙蛋白I（rhcTnI）的单克隆抗体，并对其单克隆抗体的特性进行鉴定。方法利用纯化后的rhcTn I作为免疫原，常规免疫Balb／c小鼠．取其脾细胞与同系骨髓瘤细胞Sp2／0进行常规融合。用甲基纤维素半固体培养基法获得单克隆抗体杂交瘤细胞株，利用ELISA、Western blot等方法对单克隆抗体的特性进行鉴定。结果筛选出3株稳定分泌抗rhcTnI的单克隆抗体杂交瘤细胞株，分别命名为4B3、7D5和3E6。 3株单克隆抗体的免疫球蛋白亚类分别为IgGl（4B3、7D5）和IgG2b（3E6）。3株单克隆抗体与rhcTnI发生特异性结合，而与大肠杆菌菌体蛋白、乳酸脱氢酶（LDH）、肌酸激酶（CK）、肌酸激酶同工酶（CK-MB）无特异性交叉反应，3株单克隆抗体的亲和力常数分别为1．76×10^9L／mol、1．43×10^9L/mol和1．04×10^9L/mol。结论获得了效价高、特异性好的抗rhcTnI的单克隆抗体，为cTnI诊断试剂盒的研制奠定了基础。

Preparation and identification of monoclonal antibodies against recombinant human cardiac troponin I

Objective To obtain monoclonal antibodies（mAb） against recombinant human cardiac troponin I（rhcTn I） and identify their characteristics. Methods Balb/c mice were immunized with rhcTn I. The spleen cells of the immunized mice were fused with Sp2/0 cells by regular cell fusion and hybridoma cell lines were screened by methyl cellulose semi-solid culture medium contained HAT. The characteristics of these monoclonal antibodies were determined by the techniques of ELISA, Western blot etc. Results Three hybridoma cell lines （4B3, 7D5 and 3Es） stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgGI（4B3 and 7D5） and IgG2b（3Es）, respectively. They were bound specifically to rhcTn I and failed to E. coti somatic protein, lactic dehydrogenase（LDH）, creatine kinase（CK） and CK-MB. In addition, the Kaff （affinity constant） of these three monoclonal antibodies were 1.76 × 10^9 L/mol, 1.43 × 10^9 L/mol and 1.04× 10^9 L/mol, respectively. Conclusion Monoclonal antibodies against rhcTn I with high titers and specificity is successfully prepared, which established the foundation for further study of cTn I kits.