葡萄球菌肠毒素DAS-ELISA检测方法的建立及应用  

Development and Application of DAS-ELISA for Detection of Staphylococcal Enterotoxin

作　　者：刘鹏翀[1] 黄金海[1] 刘莉[1] 刘莹[1] 刘壮[2] 孙盈[1] 

机构地区：[1]天津大学化工学院,天津300072 [2]天津市乳品食品监测中心,天津300381

出　　处：《食品科学》2012年第8期195-198,共4页Food Science

基　　金：天津市应用基础及前沿技术研究计划项目(08JCZDJC22600)

摘　　要：为筛选特异性强、亲和力高、可夹心配对单克隆抗体4A2和5C12,通过方阵试验优化工作条件,建立金黄色葡萄球菌肠毒素(staphylococcal enterotoxins,SEs)的双抗体夹心-酶联免疫吸附试验定量检测方法。结果表明:该方法标准曲线为y=4.074x-1.1888,R2=0.9892,检测下限为0.307μg/mL,批内和批间变异系数均小于10%,脱脂乳粉中SEs掺入试验测定回收率为103%～107%,特异性、重复性和稳定性良好;应用所建双抗体夹心-酶联免疫吸附试验方法,对数株葡萄球菌分离株体外培养上清中SEs产生的动态变化进行分析表明,不同菌株SEs分泌能力不同,且0～20h分泌量不断增加,对鲜牛乳和猪淋巴组织匀浆中的SEs进行检测,检出率分别为51.7%和59.1%,并与进口商品化试剂盒检测结果比对,一致率达92.5%,表明食品中SEs污染的普遍存在。所建方法灵敏高效,可为食源性污染监控提供有效手段。Staphylococcal enterotoxin（SE） is an important pathogenic factor for human food-borne diseases.Based on a couple of monoclonal antibodies with high sensitivity and affinity,a rapid,sensitive,specific DAS-ELISA method for the quantitative detection of SE was established.The linear regression equation was y=4.074x-1.1888（R2=0.9892）.The detection limit was 0.307 μg/mL.The average recovery rates for SE in skim milk powder were 103%-107% with relative standard deviation（RSD） of less than 10%.In addition,the method was also successfully used for monitoring the dynamic change of SE secretion in culture supernatants of Staphylococcus aureus isolates,indicating that it can be used not only for the detection of enterotoxin-producing strains but also for monitoring the secretion of SE.Moreover,the positive rates of the method for the detection of SE in fresh milk and porcine lymphoid samples were 51.7% and 59.1%,respectively.The total coincidence rate between the results obtained by the method and imported kit was 92.5%.Therefore,the method was sensitive and efficient and could provide an effective technical support for monitoring food-borne pollution.

关 键 词：葡萄球菌 葡萄球菌肠毒素 双抗体夹心-酶联免疫吸附法 检测方法 

分 类 号：R378.11[医药卫生—病原生物学]