Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

An indirect competitive enzyme-linked immunosorbent assay （icELISA） based on polyclonal antibody for the estrogen diethylstilbestrol （DES） was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol （DES-CP） and mono-O-carboxymethyldiethylstilbestrol （DES-CM） with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carder proteins bovine serum albumin （BSA） by mixed-anhydride method for immunogen and conjugated with ovalbumin （OVA） by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection （LLD） of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.     

农产品中的农药残留及其分析技术的发展趋向

介绍了我国蔬菜、茶叶、水果中农药残留的现状以及农产品中农药残留分析技术的研究现状和发展趋向     

Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.     

人工雌激素己烯雌酚酶联免疫分析方法的建立

本实验旨在初步建立人工合成雌激素己烯雌酚（DES）的酶联免疫吸附分析（icELISA）方法。实验首先合成了己烯雌酚的两种半抗原正丁酸己烯雌酚单醚（DES-CP）、乙酸己烯雌酚单醚（DES-CME）。DES-CP与牛血清蛋白（BSA）偶联作为免疫原免疫BALB/c雌性小鼠并进一步制备了DES单克隆抗体，表征了单克隆抗体的亲和力和特异性。在ELISA优化的条件下，方法的抑制中浓度为 IC₅₀＝9.8 ng/mL，检测限 IC₂₀＝2.3 ng/mL，工作浓度范围为 2-42 ng/mL. 抗体与己烷雌酚、双烯雌酚的交叉反应分别为 44%, 27%，与天然雌激素雌二醇的交叉反应小于0.1%. 评估了分析缓冲液中盐离子浓度、pH、有机溶剂含量等因素对分析方法的影响。本分析方法应用于水样的添加实验，回收率符合分析精度要求。HPLC法对实验结果进行了确证，证明了ELISA应用于水样分析的可靠性。