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Objective: To prepare monoclonal antibodies (mAbs) against flagellin core protein of Vibrio (V.) parahaemolyticus and establish the double-sandwich ELISA for testing V.parahaemolyticus from food products., Methods: BALB/c mice were immunized with flagellin which was extracted from V.parahaemolyticus ATCC17802 through differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells in log-phase growth when the antibody titer in serum was 1:32 000. The hybridoma cell lines were screened by regular subcloning approach and used to generate ascites. And mAbs reacting to V.parahaemolyticus flagellin were obtained by purifying from the ascites., Results: Six hybridoma cell lines secreting mAbs against V.parahaemolyticus flagellin were prepared and named VpNo.1-VpNo.6. The mAb titer in serum by indirect ELISA was 1:2×10(6);. The mAbs were subjected to Ig classes and subclasses detection and Western blotting. The Ig subclasses of these mAbs were IgG1, IgG1, IgM, IgG1, IgG2a and IgM, respectively. SDS-PAGE showed that the flagellin protein molecular weight (Mr;) was 42 000. Using VpNo.6, Double-sandwich ELISA kit was set up and applied in detecting V.parahaemolyticus, and its sensitivity was 10(3); CFU/mL. The ELISA showed VpNo.6 had a desirable specificity to V.parahaemolyticus in testing 134 V.parahaemolyticus and 74 non-V.parahaemolyticus strains. The lowest limit of detection was 21 CFU/g sample in the base-material addition test., Conclusion: The monoclonal antibodies against flagellin core protein of V.parahaemolyticus were successfully prepared. The double-sandwich ELISA we established using VpNo.6 mAb had a sensitivity as high as 10(3); CFU/mL. The monoclonal antibody of VpNo.6 was highly specific to V.parahaemolyticus.

Aim: To prepare and characterize the mouse monoclonal antibodies against Vibrio parahaemolyticus OmpW., Methods: The OmpW amino acid sequence from three diseased Vibrio was analyzed by Bioinformatics. Mice were immunized with r-OmpW which was highly expressed and purified in E.coli. Five Vibrio(Va, Vp, Vh, Vv, Van) were chosen as antigen for mAb selection.The characters of the anti-OmpW monoclonal antibodies were studied by Western blot, Flow Cytometry, indirect immunofluorescence., Results: OmpW was testified a highly conservative membrane protein.Three clones of anti-OmpW mAb was obtained. The Ig subclass of the mAb secreted from fused cell S5C10 was IgG3, which of the titer was 4.6×10(4);. The mAb could specifically recognize Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, which could not react with Pseudomonas flurosecens, Aeromonas hydrophila, Aeromonas sobria, Aeromonas hydrophila, Escherichia coli., Conclusion: The mAb could specially recognize five diseased Vibrio, which is a useful tool for the further study of the diagnosis of Vibrio.