PRODUCTION OF ANTIBODY SELECTED FOR DETECTION OF PLASMA IMMUNOGLOBULINS IN STRIPED CATFISH Pangasius hypophthalmus
Striped catfish (Pangasius hypophthalmus) is an economically relevant fish species that is primarily cultured in southeast Asia, while also becoming a common foodfish around the world. Despite its importance as a major aquaculture species, there have been few studies tailored to understand the normal physiology of this fish, especially when it comes to its immunology. The scarcity of this information often makes diagnostic and management decisions difficult when it comes to addressing fundamental issues of fish welfare. In an effort to help our understanding of humoral immune responses in striped catfish, this work aimed to isolate, purify, and characterize plasma immunoglobulins (Igs). Additionally, the purified Ig from striped catfish was used to produce a polyclonal antibody specific for this catfish species.
The first part of this study consisted of the isolation of striped catfish antibodies. Four catfish (mean weight of 340 g) were kept in a recirculating aquaculture system. Individual fish were immunized on day 0 with 195 µg of the F[ab']2 fragment of a goat IgG. On days 14 and 28, the fish received a booster with doses of 195 and 100 µg, respectively. Blood samples were collected on days 35, 42 and 49. Striped catfish Ig was separated from fish plasma by affinity chromatography. Purity of Ig was confirmed via SDS PAGE.
The second part of this project dealt with production of anti-striped catfish antibodies. To achieve this, purified striped catfish Ig were concentrated and sent to a commercial vendor that generated these antibodies in rabbits during a period of 72 days. Plasma from rabbits was received, and rabbit IgG was purified via use of protein A-affinity chromatography. Specificity of anti-striped catfish was checked via western blotting, which revealed that this antibody was selective for the heavy chain of the striped catfish Ig. Additionally, an Ouchterlony double-diffusion gel showed the lack of reactivity of this antibody towards different non-Pangasius fish species.
The third portion of this work entailed the use of the polyclonal antibody to determine total levels of plasma Ig in striped catfish. A sandwich ELISA was designed and optimized for use of striped catfish plasma (n=69). Data from this assay helped determine the normal values, also termed reference intervals, of Ig in healthy striped catfish to be in the range of 1-7 mg/mL, with a mean value of 3.5 mg/mL.
Findings from this study can help guiding decisions in striped catfish diagnostic and management cases. Moreover, the use of this technology can be also applicable for the purpose of measuring total Ig levels in other fish species.