肠炎沙门氏菌FliC单克隆抗体阻断ELISA检测方法的建立及初步应用

Establishment and preliminary application of a FliC specific monoclonal antibody-based blocking ELISA for the detection of Salmonella Enteritidis antibody

为建立肠炎沙门氏菌(SE)血清学检测方法,本研究原核表达了SE的FliC蛋白高变区,经SDSPAGE和western blot鉴定分析,结果显示,原核表达的His-FliC和GST-FliC重组蛋白分别约为52 ku和70 ku,能够与SE阳性鸭血清特异性反应。GST-FliC重组蛋白经纯化后免疫BALB/c小鼠(150μg/只),经细胞融合、克隆和筛选获得1株阳性杂交瘤细胞,命名为1D3。Western blot结果显示MAb 1D3可以特异性识别g,m型沙门氏菌鞭毛。以MAb 1D3为阻断抗体建立阻断ELISA(bELISA)方法,经条件优化后确定His-FliC重组蛋白包被浓度为4μg/mL,MAb 1D3稀释倍数为1:16000,包被条件为4℃12 h,封闭液为5%脱脂乳;利用该方法检测239份阴性鸭血清计算阻断率为30.46%(cut-off值)。采用该bELISA方法检测SE、鼠伤寒沙门氏菌、鸡白痢沙门氏菌、鸡伤寒沙门氏菌、阿培依姆沙门氏菌、大肠杆菌、阿尼蒂斯葡萄球菌、鸭疫里默氏杆菌的阳性血清,结果显示,除SE阳性血清外,该方法与其余病原阳性血清均不发生交叉反应,特异性强;利用本研究建立的bELISA方法与IDEXX试剂盒检测不同稀释倍数的SE阳性血清,结果显示,两种方法最低检测稀释倍数均能够达到1:320,敏感性较高;重复性试验结果显示,b ELISA方法的批内和批间变异系数均小于10%,重复性好。利用本实验建立的bELISA与IDEXX公司试剂盒对105份鸡血清样品和3个鸭场的48份鸭血清样品检测,结果显示,与IDEXX试剂盒比较,本实验建立的bELISA方法特异性为98.97%、敏感性91.07%,二者符合率为96.30%。利用本实验建立的bELISA方法和微量试管凝集试验(MAT)分别检测人工感染北京鸭血清抗体,两种方法结果均显示107cfu/只和106cfu/只SE感染7日龄的北京雏鸭后4 d~6 d产生抗体应答,两周后抗体效价达到峰值;109cfu/只感染14周龄北京鸭,感染后5 d抗体阳性率达到80%,感染后11 d平均抗体效价达到1:120。本研究首次利用SE FliC蛋白制备MAb建立了阻断ELISA方法,该方法具有特异性好、敏感性高、特异性强的特点,能够快速有效检测SE感染的禽血清抗体,为禽SE的流行病学调查提供技术手段。

To establish serological detection method for epidemiological investigation of Salmonella Enteritidis(SE),hypervariable region in FliC protein on SE was expressed.The result of SDS-PAGE and western blot assay revealed that His-FliC and GST-FliC recombinant protein expressed in prokaryotic cells was about 52ku and 70ku and could specifically react with SE positive duck serum.The GST-FliC recombinant protein was purified to immune mice(150μg/mouse).One hybridoma cell named 1D3 was obtained through cell fusion and subcloning selection.The result of western blot displayed that MAb 1D3 specifically bound to g,m antigen of Salmonella.A blocking ELISA(bELISA)method was established based on the MAb 1D3,and the optimal conditions were as follows:the coating concentration of His-FliC protein was 4μg/mL,the optimal dilution of MAb 1D3 was 1:16000,the coating condition was 4℃12 hours,and the blocking reagent was 5%skim milk.The cut-off percentage inhibition value was set at30.46%after testing for 239 negative duck sera using this method.The specificity detection showed that except the reaction with SE positive serum,there was no obvious cross-reaction with serum against S.Typhimurium,S.Pullorum,S.Gallinarum,S.Apeyeme,E.coli,S.Anesti,R anatipestifer,respectively.The SE positive serum was serially diluted and tested with this method and the IDEXX kit to evaluate the sensitivity of this method,the results showed that the lowest detection level of the b ELISA was 1:320dilution of positive serum,which was the same with the IDEXX kit.The repeatability test results showed the coefficient of variation of the intra-and inter-assay repeated tests were less than 10%.Comparison of the bELISA with IDEXX kit was carried out by testing 105 samples of chicken sera and 48 samples of duck sera,the results showed that the specificity and sensitivity of the bELISA were 98.97%and 91.07%,respectively,compared with IDEXX kit,and the coincidence rate was 96.30%between the two methods.Serum of ducklings infecting with 107cfu or 106cfu S.Enteritidis were collected and tested,the result revealed that antibody response generated after 4 days-6 days post infection,and the highest level reached at 14 days post infection.In addition,80%14-week-old Peking ducks challenged with 109cfu S.Enteritidis produced antibody at 5 days post infection,and the average titer of antibody reached 1:120 at 11 days after infection.Taken together,a bELISA method was developed for the first time using anti-FliC protein of S.Enteritidis MAb.The method possesses good specificity,sensitivity,and reproducibility,and is rapid and effective for detection of serum antibody against S.Enteritidis,providing a reliable means for epidemiological investigation of avian S.Enteritidis disease.