论文部分内容阅读
目的制备Cd2+单克隆抗体(McAbs,单抗),建立Cd2+间接竞争酶免疫学检测方法(ELISA)。方法利用双功能螯合剂iEDTA将Cd2+分别与血蓝蛋白(KLH)和牛血清白蛋白(BSA)偶联,制备免疫抗原和检测抗原。免疫Balb/c小鼠,应用杂交瘤技术制备抗Cd2+的单抗。鉴定单抗的效价、特异性、亚类及亲和力。以该抗体为基础建立检测镉离子的间接竞争ELISA方法,分析该方法的灵敏度、检测限、工作范围。结果成功制备出Cd-iEDTA-KLH和Cd-iEDTA-BSA偶联物;筛选出一株稳定分泌抗Cd-iEDTA单抗的杂交瘤细胞株C2C5,该细胞株分泌的抗体亚类为IgG1,腹水抗体效价为1×106,相对亲和力结合常数Ka为1.6×109L/mol。特异性结果显示,除Hg2+外,该抗体与Pb2+、Fe3+、Mn2+、Mg2+、Zn2+、Al3+、Ni2+、Ca2+、Cu2+等金属离子的交叉反应低。建立了检测镉离子的间接竞争ELISA方法,该方法的检测限为10ng/mL,IC50为250ng/mL。结论建立了镉离子的竞争ELISA检测方法,检测限达到无公害水产品的国家标准(100ng/g)。
Objective To prepare Cd2 + monoclonal antibody (McAbs) and establish indirect immunosorbent assay (ELISA) of Cd2 +. Methods Dual-functional chelator iEDTA was used to conjugate Cd2 + with hemocyanin (KLH) and bovine serum albumin (BSA) respectively to prepare immunogens and test antigens. Balb / c mice were immunized and hybridoma technology was used to prepare anti-Cd2 + monoclonal antibodies. Identify the potency, specificity, subclass and affinity of MAb. The indirect competitive ELISA method for detecting cadmium ion was established based on the antibody, and the sensitivity, detection limit and working range of the method were analyzed. The results showed that the conjugate of Cd-iEDTA-KLH and Cd-iEDTA-BSA was successfully prepared. A hybridoma cell line C2C5 stably secreting McAb against Cd-iEDTA was screened. The subclass of antibody secreted by this cell line was IgG1. The antibody titer was 1 × 106, and the relative affinity binding constant Ka was 1.6 × 109 L / mol. Specific results showed that except Hg2 +, the antibody had low cross-reactivity with Pb2 +, Fe3 +, Mn2 +, Mg2 +, Zn2 +, Al3 +, Ni2 +, Ca2 +, Cu2 + and other metal ions. An indirect competitive ELISA was developed for detecting cadmium ions. The detection limit of this method was 10 ng / mL and the IC50 was 250 ng / mL. Conclusion The competition ELISA method for determination of cadmium ion was established, and the detection limit reached the national standard (100 ng / g) of pollution-free aquatic products.