Preparation And Identification Of Monoclonal Antibodies Against Aeromonas Hydrophila And Aeromonas Sobria

Aeromonas hydrophila and Aeromonas sobria are the pathogenic bacteria that can infect various farmed and wild fish, shrimp, frog, et al and cause great economic loss in aquaculture. They can also infect people by food chain.For adapting to the development of fish culture, and for convenient detecting, curing and preventing the infection of these bacteria, We developed and identified the monoclonal antibodies(mAbs) against Aeromonas hydrophila and Aeromonas sobria. There were a series of experiments and the methods and results were as follows:1. Hybridoma technology was used to produce mAbs against Aeromonas hydrophila and Aeromonas sobria. By fusing of mouse Sp2/0 myeloma cells and spleen cells of BALB/c mouse immunized by inactivated Aeromonas hydrophila. The positive clones which produced mAbs against Aeromonas hydrophila were screened by enzyme linked immunosorbent assay(ELISA). After cloning, five strains of hybridoma named respectively 1A8, 1F11, 1G10, 3F11 and 4G10 stably secreting specific mAbs against Aeromonas hydrophila were obtained. Supernatant titers and hydroperitoneum of the antibodies were 1×10^-2～1×10^-3 and 1×10^-5～1×l0^-6 mensurating by ELISA. Identification of subclass showed that 1F11 belonged to IgG1, 1A8 belonged to IgG2a, 1G10, 3F11 and 4G10 belonged to IgG2b. Indirect ELISA detection demonstrated that 3F11 had no cross-reactivities with the other bacteria of Vibrio, showing that this mAb have high specificity and could be used to detect Aeromonas hydrophila. The mAbs against Aeromonas hydrophila might also be useful tool for studying biological properties of Aeromonas hydrophila. By fusing of mouse Sp2/0 myeloma cells and spleen cells of BALB/c mouse immunized by inactivated Aeromonas sobria. The positive clones which produced mAbs against Aeromonas sobria were screened by enzyme linked immunosorbent assay(ELISA). After cloning, six strains of hybridoma named respectively 1A8, 2A8, 2F11, 3C6, 3D11 and 4G2 stably secreting specific mAbs against Aeromonas sobria were obtained. Supernatant titers and hydroperitoneum of the antibodies were 1×10^-2～1×10^-3 and 1×10^-5～1×10^-6 mensurating by ELISA. Identification of subclass showed that 2A8 and 2F11 belonged to IgG1, 1A8 and 3D11 belonged to IgG2a, 3C6 belonged to IgG2b, 4G2 belonged to IgG3. Indirect ELISA detection demonstrated that 3C6 had no cross-reactivities with the other bacteria of Vibrio, showing that this mAb have high specificity and could be used to detect Aeromonas sobria. The mAbs against Aeromonas sobria might also be useful tool for studying biological properties of Aeromonas sobria.2. Using 10LD₅₀ of Aeromonas hydrophila challenging suckling mice, the protective properties of mAbs against Aeromonas hydrophila were studied. The results showed that the protective effect of mAb 1G10 was significant, and the protective rate of suckling mice was 62.50%. These results indicated that mAb 1G10 might be directed against neutralized epitopes of Aeromonas hydrophila and have ability to neutralize or reduce the toxicity of Aeromonas hydrophila, so it could be used to cure the infection of Aeromonas hydrophila and produce anti-idiotype mAbs against Aeromonas hydrophila. Using 10LD₅₀ of Aeromonas sobria challenging suckling mice, the protective properties of mAbs against Aeromonas sobria were studied. The results showed that the protective effect of mAb 3C6 was significant, and the protective rate of suckling mice was 62.50%. These results indicated that mAb 3C6 might be directed against neutralized epitopes of Aeromonas sobria and have ability to neutralize or reduce the toxicity of Aeromonas sobria, so it could be used to cure the infection of Aeromonas sobria and produce anti-idiotype mAbs against Aeromonas sobria.In conclusion, in this experiments we produced mAbs against Aeromonas hydrophila for the first time, selected out protective mAb 1G10. The mAb might be useful in detecting, curing of this bacteria and its infection. We produced mAbs against Aeromonas sobria for the first time, selected out protective mAb 3C6. The mAb might be useful in detecting, curing of this bacteria and its infection.