Development of monoclonal antibodies to rohu [ Labeo rohita] immunoglobulins for use in immunoassays

• Published in: Fish & shellfish immunology 2008-12, Vol.25 (6), p.761-774
• Main Authors: Rathore, Gaurav, Kumar, Gokhlesh, Sood, Neeraj, Kapoor, D., Lakra, W.S.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Carps - immunology; Catla catla; Chromatography; Clarias gariepinus; Edwardsiella tarda; Electrophoresis; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Enzyme-Linked Immunosorbent Assay - veterinary; Female; Fish Diseases - diagnosis; Fish Diseases - microbiology; Flow cytometry; Immunoglobulins; Immunoglobulins - immunology; Immunoglobulins - isolation & purification; Immunohistochemistry; Labeo rohita; Mice; Mice, Inbred BALB C; Monoclonal antibodies; Ophiocephalus striatus
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2008.02.014

Serum immunoglobulins [Ig] of rohu [ Labeo rohita] were purified by affinity chromatography using bovine serum albumin as capture ligand. The purified rohu Ig [r-Ig] had a molecular weight [MW] of 880 kDa as determined with gel filtration chromatography. The heavy chain of r-Ig had an MW of 77.8 kDa and that of light chain was 26.4 kDa in SDS-PAGE. Purified r-Ig was used for the production of two anti-rohu Ig monoclonal antibodies [D7 and H4] that belonged to subclass IgG2b and IgG1, respectively. Both the MAbs were specific to heavy chain of r-Ig as seen in Western blotting. Anti-rohu Ig MAb was used as a diagnostic reagent in ELISA and immunocytochemical assays to demonstrate its application for sero-surveillance and for immunological studies in rohu. A competitive ELISA was used to demonstrate the antigenic relatedness of r-Ig with whole serum Ig of other fish species. Cross reactivity of anti-rohu Ig MAb was observed with serum Ig of Catla catla and Cirrihinus mrigala. No reactivity to serum Ig of Ophiocephalus striatus and Clarias gariepinus was seen. Anti-rohu Ig MAb was found to be suitable for the detection of pathogen specific [ Edwardsiella tarda] antibodies in serum of immunized rohu by an indirect ELISA. In flow cytometry using D7 MAb, the mean percentage [±SE] of Ig positive cells in spleen and blood of rohu were found to be 64.85% [±2.34] and 51.84% [±2.55] of gated lymphocytes, respectively. Similarly, D7 MAb also stained 52.84% [±1.30] and 10.5% of gated lymphocytes in kidney and thymus, respectively. The anti-rohu Ig MAbs also showed specific staining of Ig bearing cells in spleen sections by the indirect immunoperoxidase test.