1. 广东省深圳市易瑞生物技术有限公司
2. 深圳市检验检疫科学研究院
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杨星星, 毕思远, 朱海, 等. 滴滴涕单克隆抗体的制备及其评价[J]. 分析测试学报, 2016,35(3):271-276.
Preparation and Evaluation of DDT Monoclonal Antibody[J]. 2016,35(3):271-276.
以滴滴涕(DDT)的特征部分为基础设计并合成了半抗原4-{4-[2,2,2-三氯-1-(4-氯-苯基)-乙基]-苯基}-丁酸(DDT-H1)、4-[4-(2,2,2-三氯-1-对甲苯基-乙基)-苯基]-丁酸(DDT-H2),并采用活泼酯法制备免疫原 DDT-H1-BSA,以及混合酸酐法制备包被原DDT-H1-OVA、DDT-H2-OVA;用DDT-H1-BSA对小鼠进行免疫,通过细胞融合、筛选、克隆等步骤得到1株能稳定分泌DDT农药抗体的单克隆细胞株。细胞株经扩大培养后,注射小鼠体内产生腹水,并将其用辛酸-硫酸铵和protein A柱子纯化出单克隆抗体。其分泌的单克隆抗体免疫球蛋白亚类为IgG1,单抗腹水的效价为1.68×105,亲和力Ka为5.238×1011L?mol-1,与其它几种代谢物有一定的交叉反应。在此基础上,利用获得的单抗研究建立DDT的间接竞争酶联免疫吸附检测法(icELISA)。结果表明,所建立的间接竞争 ELISA 方法的线性范围(IC20~IC80)为6.6~521.8ng/mL,可用于检测农副产品、环境中残留的DDT及其代谢物。
The haptens 4-{4-[2,2,2-trichloro-1-(4-chloro-phenyl)-ethyl]-phenyl}-butyric acid(DDT-H1),4-[4-(2,2,2-trichloro-1-p-tolyl-ethyl)-phenyl]-butyric acid(DDT-H2) were designed and synthesized on the basis of characteristics part of DDT,and artificial antigens DDT-H1-BSA(immmue antigen),and DDT-H1-OVA.DDT-H2-OVA(coating antigens) were prepared by coupling with the carrier proteins using active ester method and mixture anhydrides method,respectively.The monoclonal antibody(MAb) against DDT was produced by immunity DDT-H1-BSA,cell fusion, screening,cloning. The MAb was prepared from ascetic fluids of Balb/c,which was purified with saturated ammonium sulfate followed by affinity chromatography on protein A.The monoclonal antibody characterized by IgG1 isotype showed a high cross-reactivity to some metabolites.The enzyme linked immunosorbent assay(ELISA) titer of ascites was 1.68×105,and the affinity of antibody(Ka) was 5.238×1011 L?mol-1.An indirect competitive ELISA was founded base on the monoclonal antibody.The results of indirect competitive ELISA showed that the linear range of the calibration curves ranged from 6.6ng/mL to 521.8ng/mL.The method could be applied in the detection of DDT and its metabolites in agriculture products and environment.
滴滴涕单克隆抗体酶联免疫检测代谢物
dichlorodiphenyltrichloroethane(DDT)monoclonal antibodyELISAmetabolites
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