抗人心肌肌钙蛋白I单克隆抗体的制备及初步鉴定

Preparation and identification of monoclonal antibody against human cardiac troponin I

为获得可用于免疫分析的人心肌肌钙蛋白I(cTnI)的单克隆抗体,用基因重组的cTnI免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞融合,用ELISA法筛选,有限稀释法进行克隆化。经2次融合,获得12株分泌cTnI单克隆抗体的杂交瘤细胞株。ELISA法测定腹水滴度大于1∶1×105,亲和常数大于1×108L/mol;此12株单克隆抗体与人心肌组织来源的cTnI可特异性结合,与cTnT、肌钙蛋白C(TnC)、人骨胳肌肌钙蛋白I(skTnI)、人骨胳肌肌钙蛋白T(skTnT)均无交叉反应。在这12株单克隆抗体中,有3株单抗可相互进行配对。该抗体的获得,为cTnI免疫诊断试剂的研发奠定了基础。

BALB/c mice were immunized with recombinant human cardiac troponin I（cTnI）,and hybridoma cell lines secreting monoclonal antibodies（mAbs） against cTnI were screened after fusion of mouse splenic cell with Sp2/0 cells by ELISA.Cloning were performed by limiting dilution.In this way,12 positive hybridomas were obtained from 2 separate fusions,and the titers in ELISA assay of ascitic fluids and affinity constants were above 1∶1×105 and 1×108L/mol respectively.All these mAbs reacted well with cTnI from human heart tissue.And no cross-reaction could be found of each mAbs with cTnT、TnC、skTnI and skTnT.Three mAbs appeared to form a sandwich with cTnI.The successful establishment of mAbs anginst cTnI may be useful in the development of detection assay.