Rapid Detection of Monoclonal Antibody against Vibrio anguillarum by Using Conjugated Colloidal Gold

Vibrio anguillarum, strain SMW5, were inactivated by formaldehyde to be used as the antigen for immunizing Balb/c mice. Three hybridomas strains secreting monoclonal antibodies (McAb) against V. anguillarum were obtained and named as 1H9, 1C12, and 6F8 with the isotypes of IgG2a, IgM, and IgG2b, respectively. The titers of 1H9 in ascites of Balb/c mice were 1:6.4×10⁵. The polyclonal antibody was prepared against V. anguillarum, with a titer of 1:3.2×10⁵. A rapid, accurate and easy gold immunochromatographic lateral flow assay methodology (GICA) was developed for detecting V. anguillarum. The monoclonal antibody, 1H9, was conjugated with colloidal gold as a signal generator on the conjugate pad. The polyclonal antibody was used as a capture antibody for the test line (T), and the goat anti-mouse IgG antibody as the capture antibody for the control line (C) on nitrocellose membrane. A GICA test strip was assembled with an absorbing pad, a conjugate pad, and a nitrocellose membrane sprayed with the capture antibody. The sensitivity of the GICA test strip towards V. anguillarum was high with a detection limit of 6.3×10⁴cfu·mL^-1. The specificity of the assay showed no cross-reaction with 7 other aquaculture pathogens, including Aeromonas hydrophila, A. cavia, A. sobria, Edwardsiella tarda, and Streptococcus agalactiae. An accurate confirmation could be completed in 10 min for the assay. It appeared that the newly developed method using the GICA test strip was adequate for V. anguillarum detection.