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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.
Animals Antibodies, Monoclonal/biosynthesis* Antibodies, Monoclonal/immunology Carps/immunology* Enzyme-Linked Immunosorbent Assay/methods* Enzyme-Linked Immunosorbent Assay/veterinary Female Fish Diseases/diagnosis Fish Diseases/microbiology Immunoglobulins/immunology* Immunoglobulins/isolation & purification Immunohistochemistry Mice Mice, Inbred BALB C