阪崎克罗诺杆菌单克隆抗体的制备及其间接竞争-ELISA检测方法

Preparation of Monoclonal Antibodies against Cronobacter sakazakii and Development of an Indirect Competitive ELISA for Detection of This Bacterium

以阪崎克罗诺杆菌标准菌株ATCC 29004免疫BALB/c小鼠,采用杂交瘤细胞技术,经过两次细胞融合共制备出7株分泌单克隆抗体的杂交瘤细胞株(1E9、2A7、2D10、3E5、5B10、6E3、12A2)。其中2A7、2D10、3E5、12A2的效价达到了1∶256 000,6E3的效价为1∶128 000,5B10的效价为1∶64 000,1E9的效价为1∶32 000。亚型鉴定结果显示1E9的亚型为Ig G1,6E3的亚型为Ig G2b,其余抗体2A7、2D10、3E5、5B10、12A2的亚型均为IgG2a。对7株抗体进行特异性检测结果显示,2A7能与3株阪崎克罗诺杆菌(ATCC 29004、ATCC 29544、ATCC 12868)都结合,1E9能与其中两株(ATCC 29004、ATCC 12868)结合,且与其他11株类似菌和常见致病菌交叉反应结果显示7个抗体均有良好的特异性。用2A7建立的间接竞争酶联免疫吸附测定法对阪崎克罗诺杆菌的检测灵敏度可达到10~6 CFU/m L。

BALB/c mice were immunized with Cronobacter sakazakii type strain ATCC29004.Seven strains(1E9,2A7,2D10,3E5,5B10,6E3,and12A2)of hybridoma cell lines that secreted monoclonal antibodies were prepared by two cycles of cell fusion using hybridoma technique.The titers of2A7,2D10,3E5and12A2reached1:256000,the titer of6E3was1:128000,the titer of5B10was1:64000and the titer of1E9was1:32000.The results of subtype identification showed that the subtype of1E9was IgG1,the subtype of6E3was IgG2b,and the subtypes of the other antibodies2A7,2D10,3E5,5B10and12A2were all IgG2a.2A7could specifically bind to3strains of Cronobacter sakazakii(ATCC29004,ATCC29544,and ATCC12868),and1E9could specifically bind to two of them(ATCC29004and ATCC12868).The results of crossreaction with11other similar pathogens showed that7antibodies had good specificity.The sensitivity of indirect competitive enzyme linked immunosorbent assay(ELISA)using2A7reached106CFU/mL for the detection of Cronobacter sakazakii.