Antibodies recognizing both IgM isotypes in Atlantic salmon

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Identification and characterization of subpopulations of cells involved in immunological reactions against invading organisms are essential for understanding defense mechanisms against disease. In lower vertebrates like teleost fish, as opposed to mammals, immune cell subsets are still poorly defined, mostly due to the lack of appropriate working tools like antibodies and functional assays. Membrane bound molecules like immunoglobulins (Ig) serve as cell surface markers for specific cell subsets and the identification of cells relies upon the production of specific antibodies towards these molecules. The present study aimed at identifying tools to separate IgM positive (IgM+) B cells from IgM negative (IgM−) non-B cell populations using flow cytometry. Several monoclonal antibodies (mAbs), and one polyclonal antibody (pAb) to both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) IgM, either commercially available or locally produced were tested for their recognition of Atlantic salmon IgM+ cells. Leukocytes were isolated from peripheral blood (PB), spleen (S) and head kidney (HK) and stained with all mAbs and the pAb, to possibly verify the approximate number of IgM+ cells in the respective tissues in salmon. To our surprise, this seemingly simple task did not reveal similar staining patterns for all antibodies as expected, but rather large differences in the number of positively stained cells were discovered. In short, positively stained cells by each antibody ranged from below 5% to above 80% with similar ratios between the antibodies in each tissue. The three most used mAbs, 4c10, N2 and 1.14; originally produced towards rainbow trout IgM, recognize only a fraction of salmon B cells as previously shown for the 4c10 mAb binding exclusively to the IgM-A isotype. In comparison, our three novel mAbs, IgF1-3, -18 and -19, bind to both IgM-A and -B isotypes as shown using intracellular staining of 293T cells transfected with both IgM-A and -B constructs. Based on binding percentages, one of three commercially available Abs, IgH FITC from Cedarlane, may also identify both isotypes. The three new IgF1-3, -18 and -19 mAbs and potentially IgH FITC from Cedarlane, provide us with great tools enabling complete depletion or enrichment of IgM+ B cells and/or IgM− T cells in Atlantic salmon.