阪崎克罗诺杆菌双抗夹心酶联免疫吸附检测方法的建立

Establishment of a double-antibody sandwich enzyme-linked immunosorbent assay for Cronobacter sakazakii detection

为建立阪崎克罗诺杆菌(Cronobacter sakazakii)的免疫学快速检测方法,分别制备了阪崎克罗诺杆菌的特异性单抗和多抗,建立阪崎克罗诺杆菌的双抗夹心酶联免疫吸附DAS-ELISA检测方法。结果:获得4株抗阪崎克罗诺杆菌单克隆抗体阳性细胞株,分泌抗体效价分别是1∶128000、1∶32000、1∶256000和1∶256000,抗阪崎克罗诺杆菌兔多克隆抗体效价是1∶128000。建立的阪崎克罗诺杆菌DAS-ELISA检测方法,分别以抗阪崎克罗诺杆菌兔多抗和单抗作为捕获抗体和检测抗体,兔多抗的最佳稀释度为1∶4000,单克隆抗体最佳稀释度为1∶1000,灵敏性可达1×106 CFU/mL;该检测方法具有良好的特异性,与常见的6种食源性病原菌都没有交叉反应,且能够同时检测6种克罗诺杆菌属细菌;重复性结果显示,该检测方法的批内批间变异系数均小于15%,具有良好的重复性。综上,利用抗阪崎克罗诺杆菌的单抗和兔多抗成功建立了DAS-ELISA方法,该方法具有良好的敏感性、特异性和重复性,可为阪崎克罗诺杆菌的快速检测提供技术支撑。

To establish a method for rapid immunological detection of Cronobacter sakazakii,a double antibody sandwich enzyme-linked immunosorbent assay was tried in this study.The representative strains of C.sakazakii were used as mixed antigens to immunize mice.Four strains of monoclonal antibodies(McAbs)against C.sakazakii were prepared by the hybridoma technique,with the titers being 1∶128000,1∶256000,1∶32000 and 1∶256000,respectively.The titer of polyclonal antibodies(PcAbs)titers was 1∶128000.To establish the double antibody sandwich ELISA,the anti-C.sakazakii PcAbs was used as the capture antibodies and anti-C.sakazakii McAbs as detecting antibodies,respectively.The optimum dilution concentrations of PcAbs and McAbs were 1∶4000 and 1∶1000,respectively.The sensitivi⁃ty of this method was 1×106 CFU/mL,and the method had good specificity with no cross-reactivity with 6 other foodborne pathogens,and it could simultaneously detect six Cronobacter species.The results of the repetitive test showed that the intra-and inter-batch coefficients of variation of the method were less than 15%.In summary,a double-antibody sandwich ELISA method was successfully established here using anti-C.sakazakii McAbs and anti-C.sakazakii PcAbs.This method possessed good sensitivity,specificity,and reproducibility;which would be a technical support for rapid screening and analysis of C.sakazakii.