[Preparation of monoclonal antibodies against flagellin core protein of Vibrio parahaemolyticus and its activity analysis].

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.