title: Monoclonal antibody to serum immunoglobulins of Clarias batrachus and its application in immunoassays.
abstract: Serum immunoglobulins of Clarias batrachus (Cb-Ig) were purified by affinity chromatography using bovine serum albumin as capture ligand. Under reducing conditions in SDS-PAGE, Cb-Ig was composed of a heavy (H) chain (68.7 kDa) and two light (L) chains (27.4 and 26.3 kDa). Purified Cb-Ig was used to produce a monoclonal antibody (MAb) designated E4 MAb that belonged to IgG1 subclass. In Western blotting, this MAb showed binding to H chain of purified Cb-Ig and putative H chains in reduced sera of C. batrachus, Clarias gariepinus and Heteropneustes fossilis. However, no binding was observed with serum protein of Labeo rohita and Channa striata. Cross-reactivity of anti-Cb-Ig MAb was observed with serum of C. batrachus, C. gariepinus and H. fossilis in competitive ELISA. In immunoblotting of non-reduced Cb-Ig with E4 MAb, four bands assumed to be tetrameric, trimeric, dimeric and monomeric form were observed. In flow cytometric analysis of the gated lymphocytes, the number of surface Ig-positive (Ig+) cells in blood, spleen, kidney and thymus of C. batrachus was determined to be 50.1 ± 3.1, 55.1 ± 3.36, 42.4 ± 4.81 and 5.1 ± 0.89%, respectively, using E4 MAb. Ig+ cells were also demonstrated in formalin-fixed paraffin embedded tissue sections of spleen, kidney, thymus and smears of blood mononuclear cells in indirect immunoperoxidase test. The developed MAb was employed to detect pathogen-specific immunoglobulins in the sera of C. batrachus immunized with killed Edwardsiella tarda, by an indirect ELISA. This monoclonal antibody can be useful tool in immunological research and assays.
authors.affiliation: Fish Health Management Division, National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow-226002, Uttar Pradesh, India. sood_neeraj@rediffmail.com
authors.forename: Neeraj
authors.initials: N
authors.lastname: Sood
authors.forename: Dharmendra K
authors.initials: DK
authors.lastname: Chaudhary
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Singh
authors.forename: Gaurav
authors.initials: G
authors.lastname: Rathore
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005399:Fishes; D005434:Flow Cytometry; D007136:Immunoglobulins / Q000097:blood / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007136:Immunoglobulins