甲基苯丙胺半抗原设计与抗体间接ELISA检测方法的优化

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

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,通过梯度稀释法优化ELISA反应体系中抗原最佳包被浓度和最佳酶标抗体浓度(1∶2000-1∶6000)等工作条件,并进行方法的稳定性、特异性验证。结果:优化后最佳的包被缓冲液为磷酸缓冲液(phosphate buffer solution,PBS),2μg·ml-1的包被抗原在37℃2 h后4℃包被过夜为最佳包被反应条件,最佳封闭条件为5%脱脂奶粉封闭120 min,最佳酶标抗体稀释度为1∶3000(1.33μg·ml-1),最佳孵育时间为60 min,最佳底物显示时间为15 min。该方法重复性实验变异系数均在5%以内,并且只能检测出MA抗体,特异性良好。结论:本研究中的半抗原结构能诱导MA抗体的产生,并成功建立间接ELISA检测MA抗体的最佳工作条件,可为MA疫苗临床前研究奠定基础。

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.