己烯雌酚特异性抗体的制备

【目的】制备抗己烯雌酚（DES）的高亲和力特异性抗体，建立检测DES的间接竞争ELISA标准曲线，为进一步研究DES快速检测试剂盒打下基础。【方法】应用DES人工免疫抗原免疫新西兰大白兔，采集抗DES的特异性抗血清，用KCjunior软件进行4参数非线性回归，建立检测DES的间接竞争ELISA标准曲线，各药物浓度在同一批次酶标板中做4次重复，共做4个批次对标准曲线进行评价，用己烷雌酚（HEX）、双烯雌酚（DIEN）、雌二醇（E2）、乙炔雌二醇（EE2）和L-酪氨酸（Tyr）来评价抗体的特异性。【结果】5只家兔免疫产生了抗DES的特异性抗血清，其中4号家兔抗血清效价达到了1﹕4 096 000竞争抑制效果最好，工作稀释浓度为1﹕1 024 000时，线性范围在0.01～50 μg?L-1，标准曲线的IC50在0.71～1.0 μg?L-1，R2在0.9952～0.9978。获得各标准浓度下B/B0的批内变异系数在0.52%～22.48%，批间变异系数在2.64%～19.51%。交叉反应试验表明，4号家兔抗血清对与DES结构相似的人工合成二苯乙烯类雌激素药物己烷雌酚（HEX）和双烯雌酚（DIEN）的交叉反应率分别为8.5%和38.5%，与天然雌激素类药物雌二醇（E2）和乙炔雌二醇（EE2）以及L-酪氨酸（Tyr）的交叉反应均小于0.001%。【结论】本文报道的抗体特异性好、亲和力高，可满足目前中国动物性食品安全评价检测的需要，为研究组织中DES残留检测的ELISA方法及其开发DES快速检测试剂盒奠定了基础。

Development of Antibodies Against Diethylstilbestrol

Four rabbit antisera against DES with high titers (>1∶1 000 000) were obtained by immunizing rabbits with DES immunizing antigen. One of them (No.4) was used to develop an indirect competitive inhibition enzyme-linked immunoassay (Ci-ELISA) for the determination of diethylstilbestrol (DES). Four calibration curves were made by KCjunior software according to 4-parameter model. In the linear range of 0.01-50μg/L, the IC50 was 0.71～1.0μg/L，R2 was 0.9952～0.9978。The coefficients of variation intra-assay and inter-assay were 0.52%-22.48%, 2.64%-19.51% respectively．The antiserum No.4 had cross reactivity of 8.5%, 38.5% with other two synthetic anabolic HEX, DIEN respectively. But had hardly cross reactivity with native estrogen E2, EE2 and Tyr.