增强化学发光酶免疫法对猪肉中盐酸克伦特罗的检测

建立了猪肉中克伦特罗(CLB)的直接竞争化学发光酶免疫检测(dc-CLEIA)方法。通过优化离子强度、pH值确立了化学发光酶免疫法的标准曲线,优化后dc-CLEIA的检出限可达0.02μg/L。猪肉中的盐酸克伦特罗用高氯酸提取、SPE净化后绘制基质曲线,基质曲线与标准曲线吻合,说明基质影响基本消除。测定0.10、1.0、5.0μg/kg 3个水平的添加回收率,平均回收率为83%~98%,批内与批间的相对标准偏差均小于15%。进一步研究了dc-CLEIA与HPLC两种方法测定猪肉样品的相关性,结果显示两者测定结果相关性良好,r=0.964 7,说明所建立的直接化学发光酶免疫方法可用于实际样品的检测,结果准确可靠。     

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%.     

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。     

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.