Development of a polyclonal antibody for detection and sensitive quantification of immunoglobulin M-like antibody in Pangasius hypophthalmus plasma

Abstract Immunoglobulins (Igs) are an essential defense mechanism in fish, yet it is difficult to reliably quantify these components because a lack of standardization in methodology, and limited commercially available reagents. In this study, a polyclonal antibody was developed for the specific detection and quantification of IgM-like immunoglobulin in the plasma of striped catfish (Pangasius hypophthalmus). The detection probe was prepared by initial isolation and characterization of the IgM-like isotype from the fish species. An SDS-PAGE analysis of the purified striped catfish antibody revealed a heavy and a light chain with molecular weights of 70 and 25 kDa, respectively, and thus an estimated size of at least 760 kDa for the tetrameric form of this antibody isotype. This fish antibody was then utilized for immunizing New Zealand white rabbits, followed by affinity chromatography and biotinylation of the rabbit anti-pangasius Ig. The polyclonal rabbit anti-pangasius antibody was strongly reactive towards fish of the genus Pangasius, and had a weaker degree of reactivity towards two members of the genus Ictalurus. The polyclonal antibody was utilized for developing a sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of plasma levels of the IgM-like molecule in striped catfish. The reference interval (RI) for this immune analyte had a range of 1.2–7.6 mg mL−1, and a median value of 3.3 mg mL−1. The ELISA methodology presented here, although not novel, it is new for fish. Implementation of these methods will help in efforts to reliably quantify the levels of total immunoglobulin in other species of fish, without the background measurements often seen with other fish detection antibodies. Knowledge of the immunoglobulin titers is also relevant, as it is the first report of the RI for this immunological analyte in striped catfish cultured in recirculating aquaculture systems. The normal physiology findings of this study will aid in monitoring changes in the health status of striped catfish, when cultured in different systems, and when the animals undergo different types of stress events (e.g. vaccination, biological insult, chemical exposure).