人艰难梭菌毒素B抗原表位抗体的制备及其ELISA检测方法的建立

Preparation of antibodies against epitopes of Clostridium difficile toxin B and establishment of a sandwich ELISA technique

目的制备人艰难梭菌TcdB抗原表位抗体,建立针对人艰难梭菌TcdB双抗体夹心ELISA检测方法。方法通过生物信息学等方法预测人艰难梭菌TcdB蛋白的抗原表位,固相多肽合成法合成抗原并制备抗体Anti-TcdB1和Anti-TcdB2,ELISA检测效价并验证其特异性。用辣根过氧化物酶(Horseradishperoxidase,HRP)标记TcdB2抗体,建立ELISA双抗体夹心法。利用棋盘滴定法筛选一抗最佳包被浓度、最佳样品稀释倍数及最适二抗工作浓度等条件,优化ELISA检测方法并确定临界值,用已知阳性、阴性标本及重组B蛋白验证该方法的特异性、灵敏度和重复性。结果间接ELISA确定Anti-TcdB1的效价为1∶512000,抗TcdB2抗体为1∶2048000。棋盘滴定等方法确定ELISA一抗最佳包被浓度0.5μg/ml,样品最佳稀释倍数1∶2,酶标抗体最佳稀释度为1∶20000。该诊断方法特异,不与其它肠道细菌感染出现交叉反应;重组B蛋白最低检测限为32.25ng/ml;试验的重复性良好,批内和批间变异系数均小于10%。结论本研究建立的检测人艰难梭菌TcdB的ELISA双抗体夹心法敏感、特异,重复性良好,可用于鉴别产毒素B艰难梭菌。

Objectives To prepare antibodies against epitopes of Clostridium difficile toxin B(TcdB)and to establish a sandwich ELISA technique to detect TcdB fromC.difficile.Methods Bioinformatics and other methods were used to predict the epitopes of the TcdB protein of C.difficile,the antigen was synthesized using solid-phase peptide synthesis,and the antibodies Anti-TcdB1 and Anti-TcdB2 were prepared.Oxidase(horseradish peroxidase,HRP)was used to label the TcdB2 antibody,thereby establishing a method of double antibody sandwich ELISA.Checkerboard titration was used to screen conditions such as the optimal coating concentration of the primary antibody,the optimal sample dilution factor,and the optimal working concentration of the secondary antibody.ELISA detection was optimized and 30 negative samples were tested to determine the critical value.An ELISA kit was used to test different bacteria to verify the method’s specificity.TcdB was double diluted and used to develop an ELISA assay.The minimum TcdB concentration was used to determine sensitivity.The same batch and different batches of the ELISA kit were used to test negative and positive samples,and the CV%value was calculated to verify repeatability.Results Epitopes of TcdB were successfully predicted,and antibodies were prepared,Indirect ELISA indicated that the titer of Anti-TcdB1 was 1:512,000 and that of Anti-TcdB2 was 1:2,048,000.Checkerboard titration and other methods indicated that the optimal coating concentration for ELISA was 0.5μg/ml,the optimal dilution factor of the sample was 1:2,and the optimal dilution of the enzymelabeled antibody was 1:20,000.This diagnostic method is specific and did not cross-react with other intestinal bacteria.The minimum detection limit of recombinant tcdB protein was 32.25 ng/ml.The assay had good repeatability.The coefficients of variation within and between batches were less than 10%.Conclusion As established in this study,double-antibody sandwich ELISA to detect C.difficile TcdB is sensitive,specific,and reproducible,and this method can be used to identify toxin-producing C.difficile.