Short Communication Monoclonal antibody to serum immunoglobulins of Clarias batrachus and its application in immunoassays Neeraj Sood ⁎, Dharmendra K. Chaudhary, Akhilesh Singh, Gaurav Rathore Fish Health Management Division, National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow-226002, Uttar Pradesh, India abstract article info Article history: Accepted 12 September 2012 Available online xxxx Keywords: Clarias batrachus Flow cytometry ELISA Immunoglobulin Immunoperoxidase test Monoclonal antibody 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, kid- ney, 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. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Teleosts are the earliest evolutionary class to possess adaptive im- munity which is composed of humoral and cell-mediated immunity. The adaptive immune responses are mediated by actions of 2 major groups of lymphocytes classified as B and T cells. Immunoglobulins (Ig), secreted by B cells, are the major effector molecules in humoral immunity and are directed to neutralize the pathogen or tag them for removal by the immune system. Teleost B cells produce 3 different immunoglobulin isotypes; namely, IgM, IgD and IgT. The IgM is the principal player in systemic immunity and IgT appears to be the teleost Ig class specialized in mucosal immune responses (Salinas et al., 2011). Monoclonal antibodies (MAbs) specifically reacting with Ig have proven to be a powerful tool for determining the level of total and specific Ig (Tang et al., 2010; Zhan et al., 2009) and number of Ig-positive (Ig+) cells in different tissues in healthy (Li et al., 2007) infected and vaccinat- ed fish (Tang et al., 2010; Xu et al., 2011). Such monoclonal antibodies have also been used for immunolocalization of Ig+ cells in lymphoid organs (Sood et al., 2011; Tokuda et al., 2000) and have contributed greatly to improved understanding of the architecture and functioning of the fish immune system. The fish Clarias batrachus, locally known as magur, is native to Southeast Asia. The natural distribution range of the species includes India, Bangladesh, Sri Lanka, Pakistan, Myanmar, Malaysia, Singapore, Philippines, Borneo, Java and Thailand (Talwar and Jhingran, 1992). The fish can occur in all types of freshwater but are more abundant in derelict and swampy waters with high turbidity. C. batrachus is a popular and valuable food fish owing to its good taste and low fat content, and fetches a high market price. Due to aerial respiration, the fish can be traded and sold live, thereby, ensuring a fresh food product. This catfish is at increased risk of developing infections by virtue of its preferential hab- itat in bottom zones of swampy waters, where the bacterial population may be 10–20 times higher than in water column (Lewis and Bender, 1961). There are reports of occurrence of diseases and health related problems in this species (Anonymous, 1981; Kanchanakhan, 2009). MAbs have been raised to Ig of a number of teleost fish (Li et al., 2007) and most of the MAbs recognize the heavy chain of Ig (Scapigliati et al., Gene xxx (2012) xxx–xxx Abbreviations: APES, 3-aminopropyltriethoxysilane; BSA, bovine serum albumin; c-ELISA, competitive ELISA; Cb-Ig, Clarias batrachus immunoglobulins; DAB, 3‐3′ diaminobenzidine; DMEM, Dulbecco's Modified Eagle Medium; FITC, fluorescein isothiocyanate; FSC, forward scatter; HAT, hypoxanthine–aminopterin–thymidine medium; HC, heavy chain; Ig, immunoglobulins; IIPT, indirect immunoperoxidase test; LC, light chain; MAbs, monoclonal antibodies; MNC, mononuclear cells; MW, mo- lecular weight; OD, optical density; OPD, ortho-phenylenediamine; PBS-T, phosphate buffer saline with 0.05% Tween-20; SDS-PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; SSC, side scatter; TMB, 3,3′,5,5′-tetramethyl benzidine. ⁎ Corresponding author. Tel.: +91 522 2442440; fax: +91 522 2442403. E-mail address: sood_neeraj@rediffmail.com (N. Sood). GENE-37871; No. of pages: 9; 4C: 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.09.044 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Please cite this article as: Sood, N., et al., Monoclonal antibody to serum immunoglobulins of Clarias batrachus and its application in immunoassays, Gene (2012), http://dx.doi.org/10.1016/j.gene.2012.09.044