大肠杆菌F_5菌毛胶体金免疫层析检测方法的建立

Development of a colloidal gold immunochromatographic assay for detection of Escherichia coli F_5 fimbriae

为建立一种特异、稳定、敏感的检测产肠毒素大肠杆菌F5菌毛的胶体金免疫层析方法,用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗F5菌毛单克隆抗体,并喷涂在结合垫上;将纯化的兔抗F5菌毛多克隆抗体和羊抗鼠IgG包被于硝酸纤维素膜上,分别作为检测线和质控线;组装试纸条,进行敏感性、特异性、稳定性、重复性试验,并对模拟样品和临床样品进行初步检测。结果显示,对F5菌毛蛋白的最小检出量为38.2μg/mL;与大肠杆菌F4、F6、F41和F18ab菌毛无交叉反应;4℃条件下密封保存120d后仍能有效检出样品;批内、批间检测F5菌毛蛋白的结果没有差别且检测线清晰,具有良好的重复性;对14份临床样品检测的结果与间接夹心ELISA、平板凝集试验、PCR的符合率为100%。

To develop a colloidal gold immunochromatographic assay for detection of enterotoxigenic Escherichia coli F_5 fimbriae,colloidal gold particles,were prepared through deoxidization of trisodium citrate,labeled to purified anti-F5 fimbriae monoclonal antibody,and the gold probe was dispensed on the glass fiber.Purified rabbit anti-F5 fimbriae polyclonal antibody and sheep anti-mouse IgG were blotted on a nitrocellulose membrane as test line and control line,respectively.The colloidal gold immunochromatographic strip was assembled with the different accessory in regular sequence.Its sensitivity,specificity,stability and reproducibility were then evaluated.Meanwhile,the simulated samples and clinical samples were tested preliminary.The assay detection limit was 38.2μg/mL.It was confirmed by experiment that the colloidal gold immunochromatographic strip of F5 fimbriae had good sensitivity（no response to F_4,F_6,F 41 and F 18ab） and stability.The strip stored at 4℃ for 120 days could still detect the samples well.The results of detection of F_5 fimbriae using inter-and intra-batches were identical,with clear test line,and good reproducibility.14 clinical samples were examined by the colloidal gold strips,the results were identical with that of indirect Sandwich ELISA,plate agglutination test and PCR.