Development of a polyclonal antibody for detection and sensitive quantification of immunoglobulin M-like antibody in Pangasius hypophthalmus plasma
Description
Immunoglobulins (Ig) are an essential defense mechanism in fish, yet it is difficult to reliably quantify these components because a lack of standardization in the methods. 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 IgG. The polyclonal rabbit anti-striped catfish antibody was reactive only against fish of the genus Pangasius, while showing no reaction against three non-Pangasius species. 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 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 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 optical density measurements often seen with fish detection antibodies. Furthermore, the findings of this study could be of aid in monitoring changes in the health status of striped catfish undergoing different types of stress events (e.g. vaccination, biological, chemical).