| Aeromonas hydrophila is the pathogenic bacteria that can infect various farmed and wild fish, shrimp, frog, etc. This will cause great economic loss in aquaculture. It can also infect people by food chain and inflict severe losses to aquaculture. Therefore, a rapid, specific and effective way for detection and identification of A. hydrophila is extreamly important.The previous study confirmed that fish could produce the immune response mediated by immunoglobulin when confronted with some pathogens. The high purity of fish immunoglobulin is very important in studying the laws of humoral immune response, developing the rapid diagnosis of pathogens and serological test of fish based on immunoglobulins, and evaluating vaccination effect.Crucian carps (Carassius auratus gibelio) serum IgM was purified by ammonium sulfate precipitation, followed by Macro-prep high Q Cartridge and Sephacryl S-300 column chromatography. Six hybridomas secreting monoclonal antibodies (mAbs) against Crucian carps serum IgM were established by fusing SP2/0 with spleen cells from BALB/c mice immunized with the purified IgM, named A1, C2, C3, F3a, F3b and F3c. The Ig isotypes of mAbs were IgG1, IgG1, IgG2a, IgG1, IgG1 and IgM, respectively. The ELISA antibody titers of C3 and F3a were 1×103 and 1×108～1×1011 in supernatant and ascitic fluid, respectively, while the others were 1×101～1×102 in supernatant, and 1×103～4×106 in the ascitic fluids. Indirect ELISA indicated that six mAbs were specific mAbs to Crucian carps serum IgM. In the western blot, all the six mAbs recognized the band with the molecular weight of 79.2kDa. IgM were purified from the ascites by Protein A affinity chromatography and labeled with HRP. And the affinity chromatography with Sepharose 4B binding the monoclonal antibodies was developed to purify serum IgM. The SDS-PAGE result demonstrated that the purified serum IgM only consists of two bands, 79.2kD of heavy chain and 25.5 kD of light chain. The inactivated whole-cell antigen of A. hydrophila was used as the coated antigen and the monoclonal antibody to IgM of carp serum as detecting antibody, an indirect ELISA method was developed to detect the IgM levels in Crucian carps (Carassius auratus gibelio). The optimal coating concentration of the inactivated whole-cell antigen was 107cfu/ml, and the best of dilution for serum was 1:160. The optimal reaction conditions of the antigen-antibody was 37℃,1.5h; the optimal dilution and reaction condition of HRP labelled mouse anti-Crucian carps IgG were 1:200 and 37℃,1.5h. The cutoff values of 450nm of the negative and positive sera were 0.178 and 0.208. The indirect ELISA was more sensitive than slide agglutination, in which the specific antibody diluted to 1:1280 could be detected. This method was with excellent precision, the intra-assay and inter-assay coefficient of variation were both<10%. The results indicated that the IgM levels in the immunized group and the infected group were significantly higher than than in the control group. Seral IgM antibody in the immunized fish began to appear at 8～10d after immunization and reached a peak during 30-40d. In the infected group there were no antibodies at 3d after infection, began to appear in the sera at 8～10d and reached a peak during 20～30d.The competitive ELISA was developed for detecting A. hydrophila infection using the inactivated whole-cell antigen of A. hydrophila as coating antigen and rabbit antibody to whole-cell antigen as detecting antibody. The optimal concentration of the coating antigen was 107cfu/mL, the working concentrations of competing antibody and sheep anti-rabbit IgG and the optimal reaction conditions were 1:5000 and 37℃, 1.0h. While the inhibition ratio (PI) of negative control was less than 30%, the samples with above 36% of PI were regarded as positive. Approximately 66.67%,56.7%, and 30% of the 60 field sera samples were found to be positive by indirect ELISA, competitive ELISA and serum agglutination test, respectively. Competitive ELISA was a little less sensitive relative to the indirect ELISA in assessing A. hydrophila antibodies in fish, but it could accommodate samples from different fish species using a single conjugate, thus eliminating the requirement for specific anti-species conjugates needed in the indirect ELISA. Serum samples from fish (n=93) were tested by the competitive ELISA and aglutination test and the data demonstrated that the coincidence between them was 89.24%. |
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