产肠毒素大肠杆菌F4菌毛抗体间接ELISA检测方法的建立

Development of an indirect ELISA for detecting specific antibody to F4 pilus protein of enterotoxigenic Escherichia coli

为建立检测产肠毒素大肠杆菌(ETEC)F4菌毛抗体的方法,本研究以纯化的重组F4菌毛蛋白作为检测抗原建立了间接ELISA方法,并对该方法的反应条件进行了优化。确定ELISA最佳条件为:包被抗原浓度为3.5 ng/孔;5%脱脂奶封闭液封闭2 h;血清最佳稀释度为1∶3 200、血清最佳作用时间为1 h;酶标二抗的最佳作用时间为1 h。该方法对F5、F6菌毛阳性血清和猪链球菌阳性血清无交叉反应,具有良好的特异性。其组内和组间的变异系数均小于10%,具有良好的重复性。利用建立的间接ELISA方法检测血清并采用PCR方法检测其肛拭子进行符合性试验,其总符合率为96.2%。临床检测312份未经ETEC免疫的猪血清,阳性率为9.9%。表明该方法可以用于ETEC F4菌毛抗体的临床检测。

To establish an assay for detection of the antibody against enterotoxigenic Escherichia coli （ETEC）, an indirect ELISA was developed with the purified recombinant F4 pilus protein as the coating antigen expressed from E.coli. The reaction conditions were optimized as coating with 3.5 ng of recombinant F4 protein per well, blocking with the 5% skimmed milk for 2 hours, and detecting with HRP-labeled SPA in 1：5 000 dilution. It was proved that the method was specific and repeatable by cross experiment, blocking test and repeated experiment. The coincidence rate between the indirect ELISA for serum samples and PCR for the rectal swab samples was 96.2%. A total of 312 swine serum samples collected from non-ETEC immunized pigs were detected by the established indirect ELISA, the positive rate was 9.9%.