Preparation and application of IgM monoclonal antibodies in half-smooth tongue sole (Cynoglossus semilaevis).

In this study, the native IgM of half-smooth tongue sole (Cynoglossus semilaevis), an economically important marine flatfish, were purified, and used as the immunogen for preparation of monoclonal antibody (mAb). Based on the mAbs, double-antibody sandwich ELISA was constructed for the detection of IgM in C. semilaevis serum and capture ELISA were established for vaccine efficacy evaluation. Results showed that two monoclonal antibodies (mAbs) specifically targeting IgM of half-smooth tongue sole (Cynoglossus semilaevis) were successfully obtained (designated as 3A12 and 5F39). The mAbs exhibited high specificity to native C. semilaevis IgM, without cross-reactivity with IgM from other teleost species. Furthermore, a double-antibody sandwich ELISA was further established for the detection of serum IgM in C. semilaevis with the mAbs pair, which showed a linear detection range of 0.156-10 μg/mL for purified IgM, with a limit of detection (LOD) of 0.313 μg/mL. Using the developed sandwich ELISA, it was found that the IgM level in large-sized fish was significantly higher than that in small-sized fish, and immunostimulants could remarkably enhance the IgM level in fish. Moreover, the specific IgM antibody levels in the serum of C. semilaevis after immunization with inactivated Vibrio vulnificus were analyzed by capture ELISA using mAb 5F39 as the detection antibody, a time-dependent variation trend was found in the specific IgM antibody titer among the immunized group. In conclusion, the mAbs targeting C. semilaevis IgM could specifically and efficiently recognize the native IgM protein, and served as a reliable tool for the detection of serum IgM, providing essential technical support for immune status assessment and vaccine development in C. semilaevis aquaculture.