基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

Fluorescent Microspheres Lateral Flow Assay Based on Immunomagnetic Separation for Detection of S. choleraesuis

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌。待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测。每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为10~2~10~6CFU/mL的猪霍乱沙门氏菌的捕获率均大于90%,特异性好;在pH=6时,以300μg/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条。采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×10~5CFU/mL,牛奶中检出限为7.6×10~5CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍。本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景。

Immunomagnetic separation（IMS） was coupled with fluorescent microspheres lateral flow assay（FM-LFA） for rapid detection of S.choleraesuis in this study.The target bacteria were firstly enriched from sample by immunomagnetic beads（IMBs）,then eluted by heat treatment and detected by fluorescent microspheres lateral flow test strip.The IMBs was labeled with 30 μg/mg antibody,and the capture efficiency was greater than 90% against 10~2-10~6 CFU/mL of S.choleraesuis with great specificity.The immunofluorescent microspheres were prepared by coupling 300 μg of 11D8-D4 monoclonal antibody with 1 mg of fluorescent microspheres at pH 6.Monoclonal antibody 5F11-B11（2.0 mg/mL） and donkey anti-mouse Ig G（1.0 mg/mL） were sprayed on nitrocellulose membrane as test line and control line,respectively.The FM-LFA based on IMS was used to detect S.choleraesuis in PBS and milk.The limits of detection in PBS buffer and milk were 1.5×10~5CFU/mL and 7.6×10~5 CFU/mL respectively,which were 10 and 200 times lower than that of traditional fluorescent microspheres lateral flow assay,respectively.The results showed that the method,which could enrich S.choleraesuis in milk effectively,could avoid matrix interference and improve the detection sensitivity,thus had a good application prospect.