Generation and characterization of the monoclonal antibody against the heavy chain of American eel (Anguilla rostrata) IgM.

The American eel (Anguilla rostrata) is a significant aquaculture fish species in China, currently facing substantial losses due to various diseases. Limited information exists regarding the immune system of the American eel, necessitating the development of new tools to assess antibody responses following disease outbreaks or vaccinations. In this study, we cloned the constant region of the American eel IgM heavy chain (Igμ), which comprises 1326 base pairs and encodes 441 amino acid residues. Notably, transcripts of American eel Igμ were predominantly detected in the spleen, head kidney, and blood. Furthermore, we produced a monoclonal antibody (mAb) specific to the American eel IgM by immunizing mice with native IgM. Western blot analyses demonstrated that the mAb specifically reacted with the heavy chain of American eel serum IgM under reducing condition, as well as with both monomeric and multimeric forms of native IgM under non-reducing condition. Flow cytometry analyses confirmed that the mAb specifically reacted with membrane IgM on American eel B cells. Utilizing this mAb, we determined that the percentages of American eel IgM+ B cells in the spleen and peripheral blood lymphocytes were 40.86% ± 9.30% and 31.38% ± 12.62%, respectively. The mAb developed in this study is instrumental for advancing research on the humoral immune response of eels and the immune effects of vaccines, thereby promoting the development of immune prevention and control technologies for eel infectious diseases centered on vaccination.