| Fish could produce the humoral immune response mediated by immunoglobulin when confronted with some pathogens. IgM is the main immunoglobulin in fish that can elicit effective specific humoral responses against various antigens. The serum antibody titer has become a more ideal and reliable index in evaluating immunity level in aquatic animal. Therefore, it is very important for the establishment of methods to detect the IgM level in antisera.Single-chain variable fragments (scFv) is spliced with the variable region of heavy chain (VH) and the variable region of light chain (VL) from end to head by an introduced flexible polypeptide Linker. ScFv is capable of binding its target antigens with affinity similar to that of the parent monoclonal antibody. scFv has some potential advantages over full-length monoclonal antibody since they are smaller, economical and can be easily amendable to genetic manipulation.In the study, we attempted to construct scFv from the monoclonal antibody which was prepared and preserved in laboratory. The total RNA was abstracted from the hybridoma cell which was secreting anti-IgM antibody, and OligodT was employed as the reverse primer to synthesize the first chain of cDNA. VH and VL genes were amplified by PCR, and the genes were cloned into the pMD19T vector to test the sequences. Followed splicing by overlap extension, linker (Gly4Ser)4was leaded in between VH downstream and VL upstream, thereby getting the F3a-scFv gene. Then the F3a-scFv gene was recombined in the prokaryotic expression vector pET32a. After it was proved to be correct by enzyme digestion and sequencing, the recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) pLysS. SDS-PAGE analysis showed the expected protein band with the molecular weight of45kDa. The recombinant protein was highly effectively expressed with the form of inclusion body. Then the F3a-scFv protein was renaturated by4-0mol/L urea gradient. After concentrated by10kDa Millipore, the concentration of F3a-scFv was up to2.6mg/mL. The indirect ELISA and western blotting analyses demonstrated that the recombinant protein has good immunoreactivity.The inactivated whole cells of A. hydrophila was used as the coated antigen and the HRP labeled single-chain fragments variable antibody (scFv) against IgM from carp serum was used as secondary enzyme-labeled antibody, an indirect ELISA was established to detect the IgM levels in Crucian carps. The optimized reaction conditions were as followings:the optimum coating conditions were with108cfu/mL of inactivated bacterial cells overnight at4℃; the optimal dilutions of antisera was1:160;,5%skin milk was used for blocking at37℃for1.5h; the optimal reaction conditions of the antigen-antibody was37℃1.5h; the secondary enzyme-labeled antibody diluted in1:50was incubated at37℃for1h; the optimal incubation condition of substrate was28℃10min. The cutoff values of OD450nm of the positive and negative sera were0.0963and0.0831..The repetitive experiments demonstrated that coefficients of variation of inter-plate and intra-plate were1.36%-6.7%and1.2%-5.3%, respectively, showing good stability. The established ELISA was used to detect the IgM levels in the immunized group and the infected group, and the results showed that IgM in both groups appeared at10d after immunization or infection and peaked during35-40d and30-35d, respectively, and the IgM levels in the immunized group were higher than those in the infected group. |
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