甲基苯丙胺半抗原设计与抗体间接ELISA检测方法的优化

目的:提供一种有效的甲基苯丙胺(methamphetamine,MA)半抗原设计,并对MA抗体ELISA检测方法进行优化.方法:将合成的MA半抗原与破伤风类毒素(tetanus toxoid,TT)和牛血清白蛋白(bovine serum albumin,BSA)分别偶联(TT/BSA-SMA)作为免疫抗原和包被抗原,...

Design of methamphetamine hapten and optimization of indirect ELISA detection method for methamphetamine antibody

Objective:To provide an effective methamphetamine(MA)hapten design and optimize the ELISA detection method of MA antibody.Methods:The synthesized MA hapten was coupled with TT and BSA proteins(TT/BSA-SMA)as immune antigen and coating antigen respectively.The working conditions such as the optimal coating concentration of antigen and the optimal concentration of enzyme-labeled antibody(1∶2000-1∶6000)in the ELISA reaction system were optimized by gradient dilution method,and the stablity and specificity of the method were verified.Results:The best coating buffer is PBS.2μg·ml-1 coating antigen was coated at 37℃for 2 h and then stayed at 4℃overnight.This is the optimal coating reaction conditions.5%skim milk powder for 120 min is the optimal sealing solution,and the best sealing time is 120 min.Besides,the optimal enzyme-labeled antibody dilution is 1∶3000(1.33μg·ml-1),the optimal incubation time is 60 min,and the optimal substrate display time is 15 min.The repeatability of this method is within 5%,and only MA antibodies can be detected with good specificity.Conclusion:The hapten structure in this study can induce the production of MA antibodies and successfully establish the optimal working conditions for indirect ELISA to detect MA antibodies,which can lay the foundation for the preclinical research of MA vaccines.