摘要
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 prepare human cystatin C（ CysC） recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum. Methods The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established,and 52 cases of human serum samples were detected by the detection system. Results Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 μg/mL. The detection limit was 4. 598 ng/mL and IC_（50） was 0. 145 μg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples.Conclusion The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.
出处
《卫生研究》
CAS
CSCD
北大核心
2017年第4期628-632,共5页
Journal of Hygiene Research
基金
国家自然科学基金(No.J1210026
81673439)
江苏省自然科学基金(No.BK20161408)
