双抗夹心酶联免疫吸附快速检测冷鲜肉中的6种血清型沙门氏菌  

Rapid Detection of Six Salmonella Serotypes in Chilled Fresh Meat by Double Antibody Sandwich ELISA

作　　者：张帅[1] 齐颖颖[1] 张红星[1] 王怡雯[1] 谢远红[1] 刘慧[1] 

机构地区：[1]北京农学院食品科学与工程学院,食品质量与安全北京实验室,农产品有害微生物及农残安全检测与控制北京市重点实验室,北京市食品安全免疫快速检测工程技术研究中心,微生态制剂关键技术开发北京市工程实验室,北京102206

出　　处：《食品科学》2016年第16期211-215,共5页Food Science

基　　金：国家高技术研究发展计划(863计划)项目(SS2012AA101606-5,2012BAD28B02-01);北京市属高等学校高层次人才引进与培养长城学者计划项目(CIT&TCD20140315)

摘　　要：为快速检测冷鲜肉中的沙门氏菌,筛选出1株产抗6种沙门氏菌单克隆抗体的细胞株,运用产生的抗体建立酶联免疫吸附检测方法(enzyme linked immunosorbent assay,ELISA)。实验采用6种血清型致病沙门氏菌制备出混合抗原,对BALB/c小鼠及新西兰大白兔进行免疫,运用杂交瘤技术进行细胞融合,制备出抗沙门氏菌单克隆抗体以及多克隆抗体,并建立双抗夹心ELISA体系检测冷鲜肉中沙门氏菌。结果表明,成功筛选出1株能稳定分泌抗沙门氏菌的单克隆抗体杂交瘤细胞株6E7,保藏编号为CGMCC 10313以及多克隆抗体,效价分别为1∶1.28×106和1∶8.0×105;将多抗作为包被抗体吸附于96孔酶标板上,并用辣根过氧化物酶标记单抗,建立双抗夹心ELISA体系;检测模拟污染肉样中沙门氏菌,其检测限为800 CFU/g;并与其他血清型沙门氏菌、志贺氏菌、阪崎肠杆菌、大肠杆菌O157:H7、金黄色葡萄球菌及单增李斯特菌均无交叉反应,特异性良好。This study aimed to prepare antibodies specific for Salmonella and to use them establish an enzyme linkedimmunosorbent assay （ELISA） system for the rapid detection of six Salmonella serotypes in chilled fresh meat. In thisstudy, BALB/c mice and rabbits were immunized with mixed antigens from six pathogenic strains of Salmonella for theproduction of monoclonal antibodies using hybridoma technology for cell fusion. The results showed that a hybridomacell line （6E7 CGMCC 10313） which could stably secret monoclonal antibody （McAb, M23） specific for Salmonella wassuccessfully screened and the polyclonal antibody （P23） was also obtained at the same time. The titer of the prepared highlypure monoclonal and polyclonal antibodies was 1：1.28 × 10^6 and 1：8.0 × 10^5, respectively. P23 was adsorbed on 96-wellmicrotiter plates as coating antibody and M23 was labeled with horseradish peroxidase for the establishment of sandwichELISA. The limit of detection （LOD） for Salmonella in artificially contaminated meat samples was 800 CFU/g. There wasno cross-reactivity with Shigella, Enterobacter sakazakii, E. coli O157：H7, Staphylococcus aureus, Listeria monocytogenesand other Salmonella.

关 键 词：沙门氏菌 杂交瘤技术 单克隆抗体 双抗夹心ELISA 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]