抗人胱抑素C单克隆抗体制备及免疫检测体系建立

发布时间：2017-12-28 12:19

本文关键词：抗人胱抑素C单克隆抗体制备及免疫检测体系建立　出处：《卫生研究》2017年04期 　论文类型：期刊论文

【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。
[Abstract]:Objective to obtain the recombinant protein of human cystatin C (cystatin C (CysC)) and 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 National Center for Biotechnology Information (NCBI) CysC gene sequence optimization synthesis of CysC gene fragment in Escherichia coli, and expression of CysC recombinant protein, immunization of Balb/c mice after technology positive hybridoma cell lines were screened by hybridoma cell fusion, preparation of ascites were purified and detected by antibody indirect ELISA the affinity of nature, establish the competitive ELISA detection system and detection of 52 serum samples. Results 4 strains secreting monoclonal antibodies against CysC cell lines, Ab3 antibody as detection antibody and horseradish peroxidase labeled, its affinity is 4.26 * 10~6L/mol, the linear range of detection for 0.011~1.924 g/mL, the detection limit is 4.598 ng/mL, the half inhibition ratio (IC_ (50)) for 0.145 g/mL. The established competitive ELISA serum test system can accurately detect 52 serum samples. Conclusion the monoclonal antibodies with high affinity and specificity were obtained, and a reliable competitive ELISA serum detection system was established, which laid the foundation for the development of CysC rapid immunoassay kit.
【作者单位】：南京大学生命科学学院医药生物技术国家重点实验室;
【基金】：国家自然科学基金(No.J1210026,81673439) 江苏省自然科学基金(No.BK20161408)
【分类号】：R392-33
【正文快照】： 长期以来血浆尿素(Urea)、肌酐(Cr)和内生肌酐清除率(Ccr)是反映肾小球滤过率(GFR)的常用检测指标[1],但是它们在血清中的浓度容易受外界因素影响,其检测特异性和灵敏性也无法满足即时快速检测的要求。因此需要更灵敏准确的GFR检测指标。胱抑素C(cystatin C,Cys C)即半胱氨酸

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