人细胞角蛋白19片段单克隆抗体的制备和鉴定

Preparation and characterization of monoclonal antibodies against human cytokeratin 19 fragment antigen 21-1

目的制备人细胞角蛋白19片段(CYFRA21-1)的单克隆抗体(mAb),并对其免疫特性进行鉴定。方法用重组CYFRA21-1蛋白免疫BALB/c小鼠,取血清效价最高小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合,筛选出阳性杂交瘤细胞株,进行亚克隆,并鉴定分泌抗体亚型;制备小鼠腹水,用protein G从腹水中纯化出CYFRA21-1的mAb;用间接ELISA测定mAb的效价,Western blot和竞争ELISA对mAb的免疫特性进行检测,竞争ELISA对mAb的临床应用性进行评价。结果获得2G8和12G7两株能稳定分泌人CYFRA21-1mAb的杂交瘤细胞株,其分泌抗体都为轻链为κ的IgG1;两株mAb都能特异识别人血清中的CYFRA21-1,效价分别为1×10-6和5×10-7;检测134份血清样本CYFRA21-1含量的结果与北京源德生物医学工程有限公司CYFRA21-1化学发光法定量检测试剂盒检测结果的相关性分别为y=0.8167x+0.3755(r=0.9772)和y=0.8142x+0.3655(r=0.9770)。结论成功制备两株人CYFRA21-1的特异mAb,为后期人血清CYFRA21-1定量测定试剂盒的完全国产化奠定了良好的基础。

Objective To prepare monoclonal antibody （mAb） against human cytokeratin 19 fragment antigen 21-1 （CYFRA21-1） and identify the immunological characteristics of the mAb. Methods The recombinant human CYFRA21-1 protein was used as antigen to immunize BALB/c mice. The mouse spleen cells with the highest sera titer were fused with SP2/0 myeloma cells. The positive hybridoma cells were screened by indirect ELISA, and the isotypes of the mAb was detected. The hybridoma cell lines producing IgG1 were injected intraperitoneally into mouse to produce high quantities mAb. Immunoglobulin was purified from ascites using protein G affinity chromatography. The titers of prepared mAb were determined by indirect ELISA. The immunological characteristics were determined by Western blot and competitive ELISA respectively. Then clinical application study about the prepared mAb was assessed. Results Two strains of hybridoma cells （2G8 and 12G7） were generated. They could stably secrete mAb against human CYFRA21-1 which belonged to the IgG isotype and contained the ~： light chain. Two strain mAb （2G8 and 12G7） exhibited high specificity for CYFRA21-1 and did not react with the other analogues. The titers of 2G8 and 12G7 were 1 ~ 10-6 and 5 ~ 10.7 separately. The correlation of between the results of CYFRA21-1 detection in 134 serum samples by the developed competitive ELISA based on the prepared mAbs and CYFRA21-1 CLIA Test Kit （Beijing Yuande Bio-Medical Engineering Co., LTD） were y = 0.8167 × ＋ 0.3755 （r = 0.9772） and y = 0.8142 × ＋ 0.3655 （r = 0.9770） respectively. Conclusions The monoclonal antibody against human CYFRA21-1 with high titer and specificity has been successfully prepared, which provided a foundation for completing localization of quantitative detection kit of human serum CYFRA21-1.