空肠弯曲菌荧光偏振抗体检测方法的建立

摘要

To establish the fluorescent polarization assay for detection of antibody to Campylobacter jejuni,the adhesion protein PEB1A of C.jejuni was expressed and the expressed recombinant protein had good antigenicity identified by westem blot.The tracer was formed by FITC labeled C.jejuni adhesion protein PEB1A,and a fluorescence polarization antibody detection method for Cjejuni was established by determining the optimum reaction concentration,reaction time of the tracer and the dilution of serum.The results showed that the positive judgment standard of the methord was FP value ≥ 150 and the negtive standard was FP value ＜150.This method only specifically reacted with C.jejuni positive sera but had no cross-reactivity with Yersinia enterocolitica,Enterotoxigenic Escherichia coli or Staphylococcus aureus antiserum,which showed the methord had strong specificity.The minimum detection titer of the antibody was 1:80.In addition,the coefficients of variations in intra-and inter-assay were both less than 10％.The FPIA method established in this study provides a feasible method for rapid detection of antibody to C.jejuni in serum.%为建立血清中空肠弯曲菌抗体的荧光偏振检测(FPIA)方法,本研究对空肠弯曲菌的黏附蛋白PEB1A进行原核表达.Western blot鉴定显示该重组蛋白具有良好的抗原性;FITC标记空肠弯曲菌黏附蛋白PEB1A形成示踪物,通过确定示踪物的最佳反应浓度、反应时间及血清最佳稀释度,建立空肠弯曲菌的FPIA抗体检测方法.结果显示:本实验建立的空肠弯曲菌FPIA抗体检测方法的检测标准为FP值≥150mP为阳性,FP值＜150为阴性.该方法仅与空肠弯曲菌阳性血清发生特异性反应,与产肠毒素型大肠杆菌、小肠结肠炎耶尔森菌、金黄色葡萄球菌的抗血清均无交叉反应,具有较强的特异性.该方法对抗体的最低检测效价为1:80,批内和批间的重复性试验变异系数均小于10％,具有较好的重复性.表明本研究建立的FPIA方法为血清中空肠弯曲菌抗体的快速检测提供了一种可行的方法.