Production and Characterization of Monoclonal Antibodies Specific to Pangasius Catfish, Basa, and Tra

Abstract

Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

Monoclonal Antibody‐Based Sandwich ELISA for Reliable Identification of Imported Pangasius Catfish

“…for this pair (96.64%) is near 100%, indicating that although they recognize the same antigenic protein (36 kDa) their epitopes are distant from each other and they therefore suffer minimal inhibition due to their simultaneous binding of the antigen. Based on these highly favorable results, these 2 antibodies, MAb 7B8 and MAbT7E10, were selected for the new sandwich ELISA as they recognize the same antigenic protein (about 36 kDa) in both raw and heat‐treated Pangasius fish extracts, as revealed by Western blot analysis (Gajewski and others 2009), and they bind complementarily to this antigenic protein, as determined by the additivity test.…”

“…Four IgG class (IgG1 subclass) MAbs, namely, T7E10, T1G11, F7B8, and F1G11, previously developed in our laboratory (Gajewski and others 2009) were used in this study. These MAbs were raised by immunizing animals with heat‐stable crude sarcoplasmic protein extract of tra fish.…”

“…At present, there is no commercially available rapid immunoassay for fish species identification, so the development of a reliable immunoassay that could be used in the field for the rapid identification of both raw and cooked seafood would be highly desirable and is urgently needed by regulatory agencies, the seafood industry, and restaurants. In a previous study (Gajewski and others 2009), we developed 4 IgG monoclonal antibodies (MAbs) raised against heated (100 °C, 15 min) tra muscle proteins. These antibodies are either specific to Pangasius fish, basa, and tra, or cross‐reactive with all common food fish species and have the potential to be utilized in a new immunoassay for the rapid identification of the Pangasius fish, raw or cooked.…”

Application of Immunoassays for Rapid Monitor of Carbofuran Residue in Vegetables

“…The preparation of mAb was the same as described previously (He, Shen, Suo, Jiang, & Hou, 2005), and the class and subclass of the isotypes of mAb were detected using a mouse antibody isotyping kit (Pierce, Rockford, IL, USA). The specificity of mAb 3D5 was evaluated by cross-reactivity with carbofuran and its analogues in dcELISA (Gajewski, Chen, & Hsieh, 2009).…”