Characterisation of monoclonal antibodies to separate epitopes on salmon IgM heavy chain

Two types of monoclonal antibodies (MAbs) to salmon IgM heavy chain, MAb type I and II, were characterised with respect to their reaction with different enzyme fragments of IgM. Protocols were devised for trypsin, pepsin and papain digestion of salmon IgM. Trypsin and pepsin digestion yielded mainly 25, 30 ·5 kDa and 52 kDa breakdown fragments of the heavy chain after reduction and denaturation. Papain gave a predominant 15 ·2 kDa fragment and minor 52 and 30 ·5 kDa fragments. Amino acid sequence analysis of the 30 ·5 kDa heavy chain fragment identified it as domains C μ3 and Cμ4 of the Fc tail of salmon IgM. MAb type I reacted with the 30 ·5 and 52 kDa fragments of the heavy chain indicating specificity for the Fc region. From this it was concluded that these two fragments, sharing a common epitope, both belonged to the Fc tail. MAb type II reacted with the 25 kDa fragment of the heavy chain. It was concluded that this fragment was the Fab (Fd) region of the μ chain. The flow cytometry analysis supports the evidence for the separate epitope specificity of these two monoclonal antibodies. MAb type II, the anti-Fab antibody, selected IgM bearing leucocytes more efficiently than the anti-Fc antibody, MAb type I.