Abstract: The full length and truncated recombinant outer membrane protein K (OMPK) of Vibrio parahaemolyticus were purified before being used to immunize 6–8-week-old female BALB/c mice. The titer of antisera was measured by an indirect sandwich enzyme linked immunosorbent assay (ELISA). The results showed that the titer of the antiserum against the full-length OMPK recombinant protein was 128-fold higher than that of the antiserum against the truncated OMPK1 recombinant protein. Several monoclonal antibodies (mAbs) were successfully generated by using the full-length OMPK recombinant protein and were used to detect V. parahaemolyticus after being conjugated with biotin and horseradish peroxidase (HRP). Indirect sandwich ELISA results indicated that only biotin conjugated VP3 and VP16 were paired with other mAbs, showing high sensitivity and good specificity to detect various V. parahaemolyticus strains. They showed no cross reactivity with 20 strains of other bacteria. Biotinylated VP16 Fab fragment-VP3 pair showed higher sensitivity to detect V. parahaemolyticus than biotinylated VP16-VP3 pair. By this method with biotinylated VP16-VP3 pair, salmon (about 5–10 CFU/25 g) and blood clam samples were found to be positive for V. parahaemolyticus. Thus, our research successfully developed a rapid, sensitive, and convenient method for V. parahaemolyticus detection by indirect sandwich ELISA.

Key words: Vibrio parahaemolyticus; outer membrane protein K; monoclonal antibody; indirect sandwich ELISA