氟乐灵抗体的制备及其酶联免疫分析方法的建立

Preparation of Trifluralin Antibody and Development of Enzyme Linked Immunosorbent Assay

该研究针对农(水)产品中氟乐灵农药残留的问题,建立了间接竞争酶联免疫分析方法,快速检测氟乐灵农药。利用3,5-二硝基-4-氯-三氟甲苯与仲胺侧链氨基反应合成半抗原2C,采用碳二亚胺法将半抗原2C与牛血清白蛋白/卵清蛋白偶联合成完全抗原。通过免疫新西兰大白兔获得多克隆抗体Pc Ab-2C,基于间接竞争酶联免疫分析方法原理对包被原和抗体的稀释倍数、包被温度、抗体和标准品稀释液等7个因素进行系统优化,最终建立间接竞争酶联免疫分析方法。结果表明:该方法的IC_(50)为54.07 ng/mL,最低检测限为7.22 ng/mL,线性范围为12.56~282.62 ng/mL,与20种氟乐灵结构类似物进行交叉反应,只有2种药物的交叉反应率大于15%,方法特异性良好;对大豆、大豆油和鳗鱼等样品进行检测,添加回收率为89.8%~106.4%,变异系数小于10%。采用气相色谱法进行验证,添加回收率为89%~108.2%,变异系数小于10%,两种方法测定结果呈现良好的线性关系(R^(2)=0.99)。该研究所建立的方法具有灵敏度强、特异性强、准确度高等优点,适用于农(水)产品中氟乐灵农药残留的高通量快速检测。

An indirect competitive enzyme linked immunosorbent assay(ic-ELISA)was developed to rapidly detect trifluralin pesticide in agricultural or aquatic products in this paper.Hapten 2 C was synthesized by the reaction of 4-chloro-3,5-dinitrobenzotrifluoride and secondary amine side chain amino group.And then hapten 2 C was coupled with BSA or OVA to synthesize complete antigens by carbodiimide method.After that,polyclonal antibody PcAb-2 C was obtained by immunizing New Zealand rabbits.Based on the principle of indirect enzyme linked immunosorbent assay,seven factors including dilution ratio of coating antigen and antibody,coating temperature,antibody and standard diluent were systematically optimized,and finally the indirect competitive enzyme linked immunosorbent assay was developed.Under the optimized conditions,the IC_(50) of this developed ic-ELISA was 54.07 ng/mL,the detection limit was 7.22 ng/mL,and the linear range was 12.56~282.62 ng/m L.The cross reaction with 20 trifluralin analogues showed good specificity(The cross reaction rate of two drugs was more than 15%).The recoveries of soybeans,soybean oil and eels were 89.8%~106.4%,and the coefficients of variation were less than 10%.The developed method was verified by gas chromatography and the recoveries were 89%~108.2%with the coefficients of variation less than 10%.The results showed a good linear relationship(R^(2)=0.99).The above data show that the established method in this study has the advantages of high sensitivity,high specificity and high accuracy,which is suitable for the high-throughput rapid detection of trifluralin pesticide residues in agricultural(aquatic)products.