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目的获得人胱抑素C(cystatin C,CysC)重组蛋白,制备亲和力高、特异性强的单克隆抗体,建立检测人血清中CysC的竞争ELISA检测体系。方法根据美国国立生物技术信息中心(NCBI)CysC基因序列优化合成CysC基因片段,并在大肠杆菌中表达得到CysC重组蛋白,免疫Balb/c小鼠后,通过杂交瘤细胞融合技术筛选获得阳性杂交瘤细胞株,制备腹水型单抗并进行纯化,通过间接ELISA法检测抗体的亲和力等性质,建立竞争ELISA检测体系,并对52个血清样本进行检测。结果获得了4株稳定分泌CysC单克隆抗体的细胞株,将抗体Ab3作为检测抗体并进行辣根过氧化物酶标记,其亲和力为4.26×10~6L/mol,检测线性范围为0.011~1.924μg/mL,检测限为4.598 ng/mL,半数抑制率(IC_(50))为0.145μg/mL。建立的竞争ELISA血清检测体系能准确检测52个血清样本。结论获得了亲和力、特异性较高的单克隆抗体,建立了可靠的竞争ELISA血清检测体系,为CysC快速免疫检测试剂盒的研制奠定了基础。
OBJECTIVE: To obtain recombinant cystatin C (CysC) recombinant protein and to prepare a monoclonal antibody with high affinity and specificity, and establish a competitive ELISA detection system for detecting CysC in human serum. Methods According to the sequence of CysC gene from National Center for Biotechnology Information (NCBI), CysC gene fragment was optimized and expressed in E.coli. The recombinant CysC protein was obtained. Balb / c mice were immunized and screened by hybridoma cell fusion technique to obtain positive hybridomas Cell lines were prepared and purified ascitic fluid monoclonal antibody by indirect ELISA method to detect the affinity of the antibody and other properties, the establishment of competitive ELISA detection system, and 52 serum samples were detected. Results Four cell lines secreting CysC monoclonal antibodies were successfully obtained. The antibody Ab3 was detected by horseradish peroxidase and its affinity was 4.26 × 10 ~ 6 L / mol. The linear range was 0.011 ~ 1.924 μg / mL, the detection limit was 4.598 ng / mL, and the half inhibition rate (IC 50) was 0.145 μg / mL. The established competitive ELISA serum detection system can accurately detect 52 serum samples. Conclusion The monoclonal antibody with high affinity and specificity was obtained and a reliable ELISA system for competitive ELISA was established, which laid the foundation for the development of CysC rapid immunoassay kit.