Characterization of polyclonal antibodies against Japanese flounder IgM derived from recombinant IgM constant region proteins

Cell and determinant markers are important in fish immunology and have vast applications in aquaculture but the availability of such markers are quite limited. Hence, there is a need to identify and also further improve existing markers in fish. Here, we developed effective polyclonal antibodies (pAbs) targeting specific parts of the Japanese flounder (Paralichthys olivaceus) IgM constant (C) region. Recombinant proteins from the CHμ2 and CHμ3 termed IgM fragment 1 (rIgM1) and from CHμ4 termed IgM fragment 2 (rIgM2) were expressed and used to construct mouse pAb-IgM1 and pAb-IgM2, respectively. pAb-IgM1 detected both the ∼77 kDa and the ∼72 kDa heavy chains detected while pAb-IgM2 marked only the ∼77 kDa heavy chain of Japanese flounder. Both pAbs detected IgM heavy chain in immune-related tissues, heart and serum. pAb-IgM2, but not pAb-IgM1, revealed cross reactions with other fish species detecting pronounced multiple IgM bands suggesting that the CHμ4 is an important functional region in the teleost IgM molecules. Finally, the pAb-IgMs detected surface IgM+ (sIgM+) and cytoplasmic IgM+ (cIgM+) B cells in Japanese flounder kidney in vivo.