斑点免疫金渗滤法快速检测沙门氏菌O_9抗原的研究

Rapid detection of O_9 antigen of Salmonella by dot immuno-gold filtration assay

目的 利用针对沙门氏菌O9抗原单克隆抗体 3- 4 7- 0和胶体金标记亲和纯化的羊抗鼠IgG ,建立一种以微孔滤膜为固相载体 ,快速检测沙门氏菌O9抗原的斑点免疫金渗滤法 (DIGFA)。方法与结果 以沙门氏菌点膜 ,加入单抗 3- 4 7- 0 ,洗涤后再加入胶体金标记的羊抗鼠IgG ,阳性结果呈红色圆点 ,阴性结果无颜色 ,整个检测过程仅需 10min。肠炎沙门氏菌标准菌株测定该方法灵敏度为 6× 10 4CFU/点。该方法可检测所有已知含O9抗原的沙门氏菌 ,而不与其它沙门氏菌、肠杆菌科其他细菌和常见革兰氏阳性细菌反应。人工感染样品经前增菌和选择性增菌处理后即可进行瞬时检测。结论 建立了一种快速、简便、准确检测沙门氏菌O9抗原的斑点免疫金渗滤法 。

The dot immuno-gold filtration assay (DIGFA) by using monoclonal antibody 3-47-0 and colloidal gold labeled with affinity purified goat anti-mouse IgG was developed for the rapid detection of O_9 antigen of Salmonella.Bacterial suspension of Salmonella was dotted on nitrocellulose membrane, and then the monoclonal antibody 3-47-0 was added and washed later on. Subsequently, colloidal gold labeled goat anti-mouse IgG was then added. The positive result showed red colored dots, while no color was observed in the negative result. The whole course of detection required 10 minutes only, and the detection limit for S.enteritidis was 6×10~4 CFU per dot. All the known Salmonella spp with O_9 antigen could be detected by this method, but other Salmonella spp without this antigen, other enterobacteria and Gram-positive bacteria showed negative results. Artificially contaminated samples or chicken infected with S. gallinarum could be easily subjected to detection after pre-enrichment and selective enrichment. It concludes that a rapid, simple and accurate method, DIGFA, for the detection of Salmonella O_9 antigen was successfully established in the present study, thus making its potential application in human and veterinary medicine.