Abstract
Monoclonal antibodies (MAbs) against immunoglobulins are invaluable molecules with several advantages over polyclonal antibodies, including uniformity and higher specificity. They are widely used in biological research, diagnosis, and treatment. The production of this kind of MAbs often involves the purification of the desired immunoglobulin and its use as an antigen for mice immunization. Considering the challenge of purifying fish serum IgM, this study, using trout as a model, investigates a simplified method for producing monoclonal antibodies against fish serum IgM without the need for IgM purification. In this method, Balb/c mice were immunized with whole trout serum as an antigen. Then, hybridoma cells were screened using an indirect ELISA, designed to detect antibodies against maltose-binding protein (MBP) in the sera of trout. The ELISA was performed on microplates coated with MBP to which the serum of a fish immunized with MBP, hybridoma supernatants and horseradish peroxidase-conjugated anti-mouse IgG were sequentially added. After the identification of positive hybridomas, the specificity of the produced antibodies for IgM was confirmed by immunoblotting. Finally, the utility of the produced MAbs as anti-IgM in ELISA was confirmed by demonstrating the presence of antibodies against other antigens in trout sera. Therefore, the method used in this study appears to be generally applicable to produce anti-Fish IgM monoclonal antibodies and potentially anti-immunoglobulins of other animal species.