金黄色葡萄球菌A型肠毒素双抗体夹心ELISA检测方法的建立及应用

Development and application of DAS-ELISA for detection of staphylococcal enterotoxin A

【目的】建立一种快速、灵敏、特异的金黄色葡萄球菌A型肠毒素(Staphylococcal enterotoxin A,SEA)检测方法。【方法】以原核表达的可溶性重组SEA蛋白为免疫原,获得特异性强、亲和力高的单克隆抗体作为捕获抗体,同时制备抗SEA兔多抗血清作为检测抗体建立双抗体夹心ELISA(Double antibody sandwich ELISA,DAS-ELISA)检测方法。【结果】该方法对SEA的线性检测区间为2-128μg/L(y=1.102x-0.07,R2=0.994),检测下限为1.89μg/L,与SEB、SEC2和SED之间无交叉反应;鲜奶SEA人工污染试验测定回收率为94%-114%,变异系数小于10%。应用该方法对46株金黄色葡萄球菌水产品分离株和164株奶牛乳腺炎金黄色葡萄球菌分离株的体外培养上清进行检测,阳性率分别为4.4%和50.6%,表明SEA污染普遍存在。【结论】建立的DAS-ELISA方法特异性、灵敏度和稳定性好,为检测SEA的食源性污染提供了有效手段。

[Objective] The research aimed to establish a rapid, sensitive and specific method for detection of staphylococcal enterotoxin A（SEA）. [Methods] The soluble protein His-tagged SEA expressed in Escherichia coli was used as the immunogen to produce specific antibodies with high affinity. A double antibody sandwich ELISA（DAS-ELISA） was developed using anti-SEA monoclonal and polyclonal antibodies as capture antibody and detection antibody respectively. [Results] There was good linearity in the toxin range of 2-128 μg/L（y=1.102x-0.07, R2=0.994） with the detection limit at 1.89 μg/L. The assay showed no cross-reactivity with SEB, SEC2 or SED. The average recovery rates for SEA in milk ranged from 94%-114% with the variation coefficient less than 10%. In addition, the method was also successfully used to detect SEA-producing strains in culture filtrates. Of 46 Staphylococcus aureus isolates from aquatic products, 4.4% were SEA-producing, while 50.6% for 164 bovine mastitis isolates. [Conclusion] Therefore, the DAS-ELISA method was sensitive and specific, and could be used as a mean for monitoring food-borne SEA contamination.