ELISA法筛查食源性致病菌特异性抗体的影响因素探讨及其应用

Influential factors of indirect ELISA for screening specific antibodies against food-borne pathogens and their application

目的探讨食源性致病菌抗体制备中的间接ELISA筛查法的最佳工作条件并建立标准化操作流程。方法选择不同抗原包被量和包被液,设置了2个主要的孵育时间和16组孵育温度,通过比较各个不同条件的S/N比值大小从而确定最佳工作条件。结果食源性致病菌特异性抗体制备中的ELISA筛选法最佳工作条件为:尽可能选择高浓度细菌进行抗原包被,抗原包被液采用细菌培养液,37℃包被8h以上,一抗和二抗与抗原间的作用时间均为37℃孵育1h。上述ELISA筛选法也适用于蛋白质包被抗原的特异性细胞株筛选研究。结论建立了ELISA筛查法的标准流程,该方法不仅适用于菌体抗原也适用于蛋白质抗原的特异性杂交瘤细胞的筛选研究。

The study aimed to discuss the best working conditions of indirect ELISA screening method during the production of antibodies against food-borne pathogens and establish a standard operating procedure.We set four main options：different antigen content,different coating buffer,2 main incubation time and 16 groups incubation temperature.The best working conditions were attained by comparing the S/N ratio of the options using indirect ELISA.In this study,we found that high concentration of bacteria should be chosen to the fullest extent possible as coating antigen and the best coating buffer was bacterial culture medium.The incubation of antigen needed at least 8 h.The reaction time of sample serum to goat anti-mouse antibody labeled with horseradish peroxidase all was 1.0 h in this method.This study develops a method for screening of specific hybridoma against not only the somatic antigen but also the protein antigen.