Establishment of Polyclonal Antibody Indirect Enzyme-Linked Immunosorbent Assay (ELISA) and the Application in the Rapid Detection of Marine Bacteria

Abstract:

In order to rapidly detect Bacillus cereus and Vibrio alginolyticus from shrimp, we immunized the specific pathogen free (SPF) of New Zealand white rabbits with V. alginolyticus and B. cereus and collected the antisera. The titer of the polyclonal antibody was higher than 1: 2000. The antisera were used as the primary antibody and the goat anti-rabbit IgG-HRP was used as the secondary antibody in indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of V. alginolyticus and B. cereus. The optimum dilution of the antiserum was determined to be 1:10000 in indirect ELISA. The optimum coated concentration of the antigen was determined to be 106 CFU/ml. The goat anti-rabbit IgG-HRP was determined to be 1: 1000. We tested the sensitivity of the serum and the lowest detectable concentration of the two strains was 106 CFU/ml. The anti-B. cereus serum had cross reaction only with B. thuringiensis. The cross reaction of the anti-V. alginolyticus serum with other bacterial species in the genus of Vibrio showed negative results. We purified 109 strains of marine bacteria from 9 sample batches including Penaeus monodon, Fenneropenaeus chinensis and Penaeus vanmamei, and these strains were timely identified using the established indirect ELISA method. Six strains of Vibrio were detected and no Bacillus was identified.