CAJ | 학술논문

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 CYFRA21-1 mAb 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 =0.8167 x+0.3755(r=0.9772)和 y=0.8142 x+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.
목적제비인세포각단백19편단(CYFRA21-1)적단극륭항체(mAb),병대기면역특성진행감정. 방법용중조 CYFRA21-1단백면역 BALB/c 소서,취혈청효개최고소서적비장세포여소서골수류세포 SP2/0융합,사선출양성잡교류세포주,진행아극륭,병감정분비항체아형;제비소서복수,용 protein G 종복수중순화출CYFRA21-1적 mAb;용간접 ELISA 측정 mAb 적효개, Western blot 화경쟁 ELISA 대 mAb 적면역특성진행검측,경쟁 ELISA 대 mAb 적림상응용성진행평개. 결과획득2G8화12G7량주능은정분비인 CYFRA21-1 mAb 적잡교류세포주,기분비항체도위경련위κ적 IgG1;량주 mAb 도능특이식별인혈청중적 CYFRA21-1,효개분별위1×10-6화5×10-7;검측134빈혈청양본 CYFRA21-1함량적결과여북경원덕생물의학공정유한공사 CYFRA21-1화학발광법정량검측시제합검측결과적상관성분별위 y =0.8167 x+0.3755(r=0.9772)화 y=0.8142 x+0.3655(r=0.9770). 결론성공제비량주인 CYFRA21-1적특이 mAb,위후기인혈청 CYFRA21-1정량측정시제합적완전국산화전정료량호적기출.
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 κlight 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 x+0.3755 (r=0.9772) and y=0.8142 x+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.