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目的制备抗双烯雌酚(DIEN)特异性抗体,为进一步研制DIEN免疫检测试剂盒打基础。方法采用4-溴丁酸乙酯对DIEN进行活化,以活泼酯法与BSA偶联制备免疫原(DIEN-CP-BSA);经紫外扫描和飞行时间质谱扫描鉴定偶联情况;以DIEN-CP-BSA免疫Balb/c小鼠制备特异性抗体,间接(竞争)ELISA评价抗血清效价及特异性。结果本试验获得较高效价的DIEN抗血清,其效价达1∶64 000,双烯雌酚半抑制浓度(IC50)为62 ng/ml;抗血清与结构类似物己烷雌酚的交叉反应率仅为0.34%,与己烯雌酚和17-β雌二醇无交叉反应;利用该抗体建立的间接竞争ELISA检测法,双烯雌酚在10～300 ng/ml呈线性关系。结论本研究制备了抗双烯雌酚(DIEN)特异性抗体,为研究畜产品中DIEN残留及开发DIEN免疫检测试剂盒奠定了基础。
Objective To prepare Dien specific antibody and lay a foundation for further development of DIEN immunoassay kit. Methods DIEN was activated by ethyl 4-bromobutyrate (BSA) and conjugated with BSA by active ester method (DIEN-CP-BSA). Coupling conditions were identified by UV scanning and time of flight mass spectrometry. -BSA immunization of Balb / c mice to prepare specific antibodies, indirect (competitive) ELISA evaluation of antiserum titers and specificity. Results The titer of DIEN antiserum was 1:64 000 and the IC50 was 62 ng / ml. The cross-reactivity of the antiserum with the structural analogues Hexestrol The rate was only 0.34%, and diethylstilbestrol and 17-β-estradiol no cross-reaction; indirect competitive ELISA assay established by the antibody, diethylstilbestrol in the 10 ~ 300 ng / ml showed a linear relationship. Conclusion DIEN-specific antibodies were prepared in this study, which laid the foundation for the study of DIEN residues in animal products and the development of DIEN immunoassay kit.