动物尿液中苯乙醇胺A胶体金快速检测方法的建立

Establishment of a Detection Method for the Immune Colloidal Gold of Phenylethanolamine A in Animal Urine

【目的】苯乙醇胺A为一种新型违禁添加剂,目前的检测方法繁琐耗时,故建立动物尿液中苯乙醇胺A胶体金免疫层析快速检测方法。【方法】通过苯乙醇胺A与溴代戊醛发生亲核取代反应,制备得到苯乙醇胺A半抗原,将苯乙醇胺A半抗原与载体蛋白偶联得到免疫原和包被原,经TNBS法测定半抗原与载体蛋白偶联比。用免疫原免疫BALB/c小鼠,制备特异性单克隆抗体,采用间接竞争ELISA方法测定单克隆抗体的灵敏度。选取与苯乙醇胺A结构类似的13种同类药物利用间接竞争ELISA方法进行单克隆抗体特异性的测定,计算交叉反应率。并通过胶体金、单克隆抗体-胶体金标记物、结合物释放垫、反应膜、样品吸收垫的制备以及试纸条的组装,建立了动物尿液中苯乙醇胺A胶体金快速检测方法,测定试纸条的检测限、假阳性率、假阴性率以及与ELISA方法的检测猪尿样品结果比较,验证试纸条的准确度,其中ELISA方法的标准品浓度分别为0、0.1、0.3、0.9、2.7、8.1μg·L-1,对猪尿样品的最低检测限为0.5μg·L-1,回收率为75%—105%。【结果】苯乙醇胺A半抗原制备结果显示从苯乙醇胺A的氨基端引入末端代醛基的连接臂,得到苯乙醇胺A衍生物,即苯乙醇胺A半抗原。TNBS法测定结果显示苯乙醇胺A-BSA和苯乙醇胺A-OVA成功偶联,苯乙醇胺A半抗原-BSA偶联比为11.38,苯乙醇胺A半抗原-OVA偶联比为10.66。间接ELISA检测结果显示MC-73杂交瘤细胞株的上清效价为1﹕2×103,腹水效价为1﹕5×107,较MC-33和MC-29的效价高,抗苯乙醇胺A单克隆抗体对苯乙醇胺A的IC50为0.365μg·L-1,较MC-33和MC-29的IC50低,得到MC-73单克隆抗体腹水灵敏度最高。苯乙醇胺A单克隆抗体与克仑特罗、莱克多巴胺和沙丁胺醇等共13种苯乙醇胺A的同类化合物的交叉反应率均小于1%,试纸条检测限为5μg·L-1,假阳性率为0,假阴性率为0。检测实际动物尿液样品时,试纸条与ELISA测定结果没有差异。【结论】成功研制出灵敏、特异、简便、快速的苯乙醇胺A胶体金免疫层析试纸条,可直接检测动物尿液,无需样品前处理,反应只需5 min即可得到检测结果。适合中国基层兽药检测实验室和企业大批量样品集中筛查及现场检测。

【Objective】 The aim of the study is to establish a detection method for the immune colloidal gold of phenylethanolamine A in animal urine. 【Method】Phenylethanolamine A hapten was synthetized by nucleophilic substitution with phenylethanolamine A and bromo aldehyde. Phenylethanolamine A hapten was coupled with BSA and OVA, respectively, to prepare immunogen and envelope antigen. The coupling ratio of hapten-BSA/OVA was determined by TNBS. Monoclonal antibody was acquired from mice which were immunized with hapten-BSA. The indirect ELISA method has been established for sensitivity and specificity detection of monoclonal antibody. A total of 13 compounds whose structures were similar with phenylethanolamine A were used to test the specificity and calculate the cross reaction rate of monoclonal antibody. A rapid detection method for immune colloidal gold of phenylethanolamine A in animal urine was established by the parts of strip prepared and assembled. The strip＇s detection limits, false positive rate and false negative rate were determined. Compared the immunochromatography assay（ICA） with ELISA whose limited ranges of standards＇ concentration were 0, 0.1, 0.3, 0.9, 2.7 and 8.1 μg·L^-1, the detection limits for pig urine was 0.5 μg·L^-1and the ranges of percent recovery were 75%-105%.【Result】Phenylethanolamine A derivative was prepared that amino terminal of phenylethanolamine A connected with the connecting arm whose aldehyde group was on the end. Phenylethanolamine A hapten connected with BSA and OVA successfully by TNBS and the coupling ratio between hapten with BSA and OVA were 11.38 and 10.66, respectively. The titer and ascites of MC-73 hybridoma cell strain were 1﹕2×10^3 and 1﹕5×10^7which were higher than MC-33 and MC-29. IC50 of monoclonal antibody against phenylethanolamine A was 0.365 μg·L^-1 which was more sensitive than MC-33 and MC-29. The cross reaction rate with 13 compounds whose structures were similar with phenylethanolamine A were less than 1%. The detection limit, false positive rate and false negative rate were 5 μg·L^-1, 0 and 0, respectively. Detection results of phenylethanolamine A in pig urine between ICA and ELISA were the same.【Conclusion】The phenylethanolamine A strip with high sensitivity, specificity, accuracy and rapidity was developed in this study which would be used to test animal urine in 5 min without pre-treatment of urine samples. It would be efficient for mass samples screening in primary laboratory and enterprises.