中国农业科学Issue(19)：3931-3940,10.DOI:10.3864/j.issn.0578-1752.2015.19.015
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
摘要
Abstract
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 8.1μg·L-1, the detection limits for pig urine was 0.5μg·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 1﹕2×103 and 1﹕5×107 which were higher than MC-33 and MC-29. IC50 of monoclonal antibody against phenylethanolamine A was 0.365μg·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 5μg·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.关键词
苯乙醇胺A/胶体金免疫层析/快速检测试纸条/动物尿液Key words
phenylethanolamine A/colloidal gold immunochromatographic assay/rapid detection strip/animal urine引用本文复制引用
聂雯莹,罗晓琴,李金超,李行,吴广,王琳琛..动物尿液中苯乙醇胺A胶体金快速检测方法的建立[J].中国农业科学,2015,(19):3931-3940,10.基金项目
国家“十二五”科技支撑计划 （）
