Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells

Abstract

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ± 1.58%, 33.00 ± 1.64%, and 16.50 ± 2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 μm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ± 0.58%, 4.053 ± 0.62% and 23.17 ± 2.26%, respectively, while only 2.36 ± 0.23%, 1.16 ± 0.44% and 6.41 ± 0.45 of IgM+ B cells in these three tissues ingested 1 μm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.