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

摘要

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 CH mu 2 and CH mu 3 termed IgM fragment I (rIgM1) and from CH mu 4 termed IgM fragment 2 (rlgM2) were expressed and used to construct mouse pAb-IgM1 and pAb-IgM2, respectively. pAb-IgM1 detected both the similar to 77 kDa and the similar to 72 kDa heavy chains detected while pAb-IgM2 marked only the similar to 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 CH A 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.