Biochemical characterization and mucosal immune function of IgT in grass carp (Ctenopharyngodon idella)

Immunoglobulin T (IgT) is a teleost-specific immunoglobulin discovered to be specialized in mucosal immunity. For fish that live in water throughout their lives, eliciting a local mucosal immune response through immersion immunization is an important and highly suitable way for preventing fish diseases. However, due to the lack of corresponding antibodies against IgT, studies on its immune responses to pathogens and vaccines in commercial fish have largely been limited to rainbow trout. Thus, a valuable tool for assessing mucosal immune response to immersion vaccination is still lacking in most commercial fish species. In this study, we expressed and purified recombinant protein of the heavy chain constant domains (CH) 1-2 of grass carp IgT and immunized mice to develop a mouse anti-grass carp IgT monoclonal antibody (mAb). Western blotting results showed that the mAb could specifically recognize grass carp IgT without cross-reacting with grass carp IgM, IgMT1 or IgMT2. We then measured IgM and IgT concentrations in the serum and gill mucus of grass carp and examined the responses of specific IgM and IgT to immersion immunization with grass carp reovirus genotype Ⅱ (GCRV-Ⅱ) and Flavobacterium columnare. We found that the concentration of IgT in gill mucus was higher than that in serum, and the IgT/IgM ratio in gill mucus was much higher than that in serum. Moreover, after immersion immunization, the specific IgT levels in gill mucus increased significantly, while no specific IgT was detected in serum. In contrast, the specific IgM levels in serum increased significantly, whereas there was little change in specific IgM levels in gill mucus. These results indicated that IgT is an important immunoglobulin in mucosal immune response, whereas IgM plays an important role in systemic immune response in grass carp. In summary, these findings not only further elucidate the biochemical and functional differences between IgM and IgT in grass carp, but also provide a valuable detection tool for accurately evaluating the immune efficacy of immersion vaccines in grass carp.