Fluorescent Microspheres Lateral Flow Assay Based on Immunomagnetic Separation for Detection of S.choleraesuis / 分析化学

ABSTRACT

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 102-106 CFU/mL of S.choleraesuis with great specificity.The immunofluorescent microspheres were prepared by coupling 300 μg of 11 D8-D4 monoclonal antibody with 1 mg of fluorescent microspheres at pH 6.Monoclonal antibody 5F11-B11 (2.0 mg/mL) and donkey anti-mouse IgG (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 1.5×105 CFU/mL and 7.6×105 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.