阪崎肠杆菌多克隆抗体的制备与鉴定

为制备高效价的阪崎肠杆菌单克隆抗体,分别以福尔马林灭活的阪崎肠杆菌(Cronobacter sakazakii)菌体和菌体破碎细胞为免疫原免疫雌性清洁级大白兔,制备抗C.sakazakii多克隆抗体,获得的抗体效价分别为256000和64000,选取菌体免疫原制备抗体。采用辛酸-硫酸铵法纯化抗体,抗体纯度用变性聚丙烯酰胺凝胶电泳鉴定。斑点杂交法检测抗体特异性,所获抗体与S.aureus, L.monocytogenes, S.typhimurium, S.flexneri, E.coli无交叉反应,与沙门氏菌有轻微交叉反应,采用杂菌抗原反向吸附法去除了该交叉反应。选取菌体免疫原免疫动物,杂菌抗原反向吸附法纯化制备获得了对C.sakazakii特异性高的多克隆抗体,为阪崎肠杆菌免疫学检测奠定了基础。

Preparation and Identification of Cronobacter sakazakii Polyclonal Antibody

To obtain high titerCronobacter sakazakii antibody,C. sakazakii polyclonal antibody was prepared in rabbits using formalin- inactivated C. sakazakii and C. sakazakii cell debris as antigen. The titers of antiserum were 256000 and 64000, respectively, and formalin-inactivatedC.sakazakii antigen was chosen to prepare the antibody. Antibody was purified by octanoic acid-ammonium sulfate precipitation method, the purity of the pAb was detected by SDS- PAGE. The antibody had no cross reaction with S.aureus , L. monocytogenes ,S.typhimurium ,S.flexneri andE.coli by dot blot hybridization method butsalmonella . To get rid of the disturb ofsalmonella , the purified pAb was dealt withsalmonella bacterial antigens with reverse adsorption method to eliminate the cross reaction withsalmonella . The high-titer pAb special toC.sakazakii could be obtained by formalin-inactivated antigen and purified by reverse adsorption method and it was a good foundation for immune detection method development.