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目的:制备PCB77人工抗原和多克隆抗体,为建立其免疫学检测方法提供技术准备。方法:以3,4-二氯苯基乙酸为半抗原,在低温和弱碱性条件下,采用N-羟基琥珀酰亚胺活性酯法将其与载体BSA偶联,制备PCB77的人工抗原,计算结合比为23∶1,免疫家兔获得了PCB77的多克隆抗体。采用混合酸酐法将半抗原与OVA偶联,结合比为8∶1,用作ELISA检测包被原。间接ELISA检测结果表明,2只兔子的抗血清效价均达1∶20 000以上。抗血清经辛酸-硫酸铵沉淀法纯化后通过ELISA检测验证了PCB77人工抗原的有效性,通过方阵滴定法确定ELISA的最佳工作浓度,建立标准曲线。在0.75~300 ng/L范围内,抑制率与PCB77的质量浓度对数呈显著的线性关系,检测限达到4.44μg/L。结论:合成的PCB77人工抗原具有较好的免疫效果,纯化后的抗体符合后续实验的条件要求,为研制和开发PCB77免疫检测试剂盒奠定了基础。
OBJECTIVE: To prepare PCB77 artificial antigen and polyclonal antibody and provide technical preparation for establishing its immunological detection method. METHODS: 3,4-Dichlorophenylacetic acid was used as a hapten and conjugated to carrier BSA by N-hydroxysuccinimide active ester method under low temperature and weak alkaline conditions to prepare artificial antigen of PCB77. Polyclonal antibodies against PCB77 were obtained from immunized rabbits by calculating the binding ratio of 23: 1. The hapten was coupled to OVA using a mixed acid anhydride method with a binding ratio of 8: 1 for detection of envelop by ELISA. The results of indirect ELISA showed that the antiserum titers of two rabbits reached more than 1:20 000. The antiserum was purified by octanoic acid-ammonium sulfate precipitation and tested by ELISA to verify the effectiveness of PCB77 artificial antigen. The optimal working concentration of ELISA was determined by square matrix titration and the standard curve was established. In the range of 0.75-300 ng / L, there was a significant linear relationship between the inhibition rate and the logarithm of PCB77 concentration, with a detection limit of 4.44 μg / L. Conclusion: The synthetic PCB77 artificial antigen has a good immune effect. The purified antibody conforms to the requirements of subsequent experiments and lays the foundation for the development and development of PCB77 immunoassay kit.