| Vibrio parahaemolyticus is one kind of intestinal bacterial which can cause serve diarrhea or other death symptom. At present, the detection methods of Vibrio parahaemolyticus are as follows:conventional detection method, immunoassay, molecular detection method and instrument detection method. The conventional detection method is accurate, but it costs long time. The molecular detection method is fast, sensetive and specific, but it needs specific knowledge. The instrument detection method is fast and accurate, but the instrument is expensive. As immunoassay is easy, fast and sensitive, it is now widely used in the detection of pathogens. In this study, polyclonal antibody and monoclonal antibody of Vibrio parahaemolyticus were prepared. The reaction condition of indirect ELISA was optimed. In addition, colloidal gold was also prepared and coupled with polyclonal antibody. Immunochromatographic test strip was also prepared. It could be used in the rapid detection of Vibrio parahaemolyticus.1. Preparation and purification of polyclonal antibody against Vibrio parahaemolyticusVibrio parahaemolyticus mode strain ATCC17802 was used as the immunogen. After 2 h of boiling water bath, the bacterial immunogen were obtained to immune rabbit. After certain immune procudure, polyclonal antibody of Vibrio parahaemolyticus was obtained. After bitter ammonium sulfate purification, the titer of the polyclonal antibodiy was 1:25600. The protein concentraton of the polyclonal antibodiy was 8 mg/mL. The molecular mass of the polyclonal antibodiy antibody was 156 kD.2. Optimation of indirect ELISAThe optimal reaction conditions of indirect ELISA were optimed. The optimal coating condition was carbonate buffer,4℃, overnight. The optimal blocking condition was 5% skim milk,37℃,0.5 h. The optimal dilution of polyclonal antibody was 1:3200, 37℃,1 h. The optimal dilution of HRP was 1:2000,37℃,1 h. The optimal substrate time was 10 min. In the optimal reaction condition, polyclonal antibody of Vibrio parahaemolyticus only reacted with Vibrio parahaemolyticus. The detection limit was 107 cfu/mL. In one ELISA reaction.3. Preparation and purification of Vibrio parahaemolyticus monoclonal antibodyThe same antigen was used to immune Bal b/C and monoclonal antibody was obtained. After octanoic acid extraction purificasation, the titer of the monoclonal antibodiy was 1:400. The molecular mass of the monoclonal antibodiy antibody was 156 kD.4. Preparation of colloidal gold and gold-labeled antibodyColloidal gold was produced with sodium citrate method.40 nm colloidal go(?) solution was got. Through Differential centrifugation, polyclonal antibody of parahaemolyticus was coupled with colloidal gold successfully.5. The preparation of immunochromatography test strip for Vibrio parahaemolyticusImmunochromatography test strip was prepared with polyclonal antibody and monoclonal antibody. The optimal materials for immunochromatography test strip were glass fiber membranes 8964 and nitrocellulose membranes Sartorius CN 140. Control lines and test line were coated with 1 mg/mL goat anti-rabbit IgG and 1 mg/mL monoclonal antibody. The test strip showed good specificity. The detection limit was 106 cfu/mL. After 3 months in room temperature in dark, the test strip showed good stability. |
