Iranian Veterinary Journal

Iranian Veterinary Journal

A Simple Method for the Production of anti-Fish IgM Monoclonal Antibodies

Document Type : Research Paper

Authors
Masoud Reza Seyfi Abad Shapouri 1
Roya Rahnama 2
Rahim Peyghan 3
Anahita Rezaie 1
Nastaran Shahbazian 4
1 Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
2 PhD Graduated of Aquatic Animal Health, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
3 Professor, Department of Livestock Animal, Poultry and Fish Health, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran and Member of Excellence Center of Warm Water Fish Health, Shahid Chamran University of Ahvaz, Ahvaz, Iran
4 Assistant Professor, Department of Aquaculture Sector, Iranian Veterinary Organization, Tehran, Iran
10.22055/ivj.2025.505300.2775
Abstract
Immunoglobulin M (IgM) is the predominant antibody isotype in the serum of teleost fish and plays a central role in systemic immune responses. Monoclonal antibodies (mAbs) targeting fish IgM are essential tools for immunological research and disease management in aquaculture. However, conventional mAb production protocols require purified IgM, which is difficult to obtain due to the complexity of purification procedures in teleost species. This study introduces a simplified method for generating anti-IgM mAbs by immunizing mice with whole serum from rainbow trout (Oncorhynchus mykiss), thereby eliminating the need for IgM purification. To do this, hybridomas were screened using an indirect ELISA based on an unrelated antigen (e.g., maltose-binding protein) and trout sera containing antibodies against this antigen. This approach led to the identification of five stable hybridoma clones (1D4, 3D3, 3E3, 4B3, and 7F6). The resulting mAbs effectively recognized trout antibodies against Infectious pancreatic necrosis virus, Lactococcus garvieae bacterium and sheep red blood cells. Immunoblotting under reducing and non-reducing conditions confirmed the specificity of these mAbs for trout IgM. This method provides a practical and efficient alternative for producing functional monoclonal antibodies against fish IgM.
Keywords
Monoclonal antibody
Trout
IgM
ELISA
Immunoblotting
Subjects
Al-Harbi, A.H., Truax, R., and Thune, R. L. (2000). Production and characterization of monoclonal antibodies against tilapia Oreochromis niloticus immunoglobulin. Aquaculture, 188(3–4), 219-227.
Ausubel, F. M. (1992). Short protocols in molecular biology, Greene Pub. Associates, Wiley edition.
Coscia, M.R., and Oreste, U. (1998). Antarctic Fish Immunoglobulins: Preliminary Data on Structure and Antibody Specificity, In book: Fishes of Antarctica: A Biological Overview, Springer/Sci-Tech/Trade, pp.175-184.
Deluca, D., Wilson, M., and Warr, G. W. (1983). Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. European Journal of Immunology, 13(7), 546-551.
Edholm, E.S., Bengtén, E., Stafford, J.L., Sahoo, M., Taylor, E.B., Miller, N.W., and Wilson, M. (2010) Identification of two IgD + B cell populations in channel catfish, Ictalurus punctatus. Journal of Immunology, 185, 4082–4094.
Elcombe, B.M., Chang, R.J., Taves, C.J., and Winkelhake, J.L. (1985). Evolution of antibody structure and effector functions: comparative hemolytic activities of monomeric and tetrameric IgM from rainbow trout, Salmo gairdnerii. Comparative Biochemistry and Physiology B, 80(4), 697-706.
Estevez, J., Leiro, J., Santamariana, M.T., Damainguez, J., and Ubeira, F.M. (1994). Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays. Veterinary Immunology and Immunopathology, 41, 353–366.
Hikima, J., Jung, T.S., and Aoki, T. (2011). Immunoglobulin genes and their transcriptional control in teleosts. Developmental and Comparative Immunology, 35, 924–936.
Huang, Y., Yuan, X., Mu, P., Li, Q., Ao, J., and Chen, X. (2019). Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells. Fish and Shellfish Immunology, 91, 216-222.
Ji, J.F., Hu, C.B., Shao, T., Fan, D.D., Zhang, N., Lin, A.F., Xiang, L.X., and Shao, J.Z. (2020). Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model. Immunology, 162(1), 105-120.
Jingzhuang, Z., Liming, X., Miao, L., Yongsheng, C., Jiasheng, Y., Hongbai, Y., Hongbai, L., and Tongyan, L. (2014). Expression and rabbit antisera preparation of IgM heavy chain gene in rainbow trout (Onchorhynchus mykiss). Journal of Fisheries of China, 38(8), 1175-1181.
Jones, E.M., Oliver, L.P., Ma, J., Leeuwis, R.H.J., Myrsell, V., Arkoosh, M.R., Dietrich, J.P., Schuster, C.M., Hawkyard, M., Kurt Gamperl, A., Kenneth, D., and Cain, K.D. (2022). Production of a monoclonal antibody specific to sablefish (Anoplopoma fimbria) IgM and its application in ELISA, western blotting, and immunofluorescent staining.  Fish and Shellfish Immunology, 130, 479-489.
Magnadottir, B. O., Kristjansdottir, H., and Gudmundsdottir, S. (1996). Characterisation of monoclonal antibodies to separate epitopes on salmon IgM heavy chain. Fish and Shellfish Immunology, 6(3), 185-198.
Mahmoodi, P., Seyfi Abad Shapouri, M.R., Ghorbanpour, M., Haji Hajikolaei, M.R., Lotfi, M., Pourmahdi Boroujeni, M., and Daghari, M. (2015). Development of a simple indirect ELISA, based on prokaryotically expressed recombinant MBP-NS3 protein, for detection of antibodies against bovine viral diarrhea virus. Jundishapur Journal of Microbiology, 8(3), e14311.https://doi.org/10.5812/jjm.14311.
Mashoof, S., Pohlenz, C., Chen, P.L., Deiss, T.C., Gatlin, D. 3rd., Buentello, A., and Criscitiello, M.F. (2014). Expressed IgH mu and tau transcripts share diversity segment in ranched Thunnus orientalism. Developmental and Comparative Immunology, 43, 76–86.
Morrison, R.N., and Nowak, B.F. (2002). The antibody response of teleost fish. Seminars in Avian and Exotic Pet Medicine, 11(1), 46-54.
Oliver, L.P., Bruce, T.J., Ma, J., Jones, E.M., and Cain, K.D. (2023). Development of a monoclonal antibody specific to burbot (Lota lota) IgM and optimization of an ELISA to measure anti-Aeromonas sp. antibody titers following pathogen challenge. Fish and Shellfish Immunology, 137, doi: 10.1016/j.fsi.2023.108775.
Parra, D., Takizawa, F., and Oriol Sunyer, J. (2013). Evolution of B Cell Immunity. Annual Review of Animal Biosciences, 1, 65-97.
Pettersen, E.F., Fyllingen, I., Kavlie, A., Maaseide, N.P., Glette, J., Endresen, C., and Wergeland, H.I. (1995). Monoclonal antibodies reactive with serum IgM and leukocytes from Atlantic Salmon (Salmo salar L). Fish and Shellfish Immunology, 5, 275–287.
Rahnama, R., Seyfi Abad Shapouri, M.R., Peyghan, R., Rezaie, A., and Shahbazian, N. (2018). Cloning and expression of the constant region of rainbow trout (Onchorhynchus mykiss) µ immunoglobulin chain in Escherichia coli. Iranian Journal of Fisheries Sciences, 17(3), 573-584.
Salinas, I., Fernandez-Montero, A., Ding, Y., and Oriol Sunyer, J. (2021). Mucosal immunoglobulins of teleost fish: A decade of advances.  Developmental and Comparative Immunology, 121, https://doi.org/10.1016/j.dci.2021.104079.
Sanchez, C., Coll, J., and Domínguez, J. (1991). One-step purification of the major rainbow trout immunoglobulin. Veterinary Immunology and Immunopathology, 27(4), 383-391.
Sanchez, C., Lopez-Fierro, P., Zapata, A., and Dominguez, J. (1993). Characterization of monoclonal antibodies against heavy and light chains of trout immunoglobulin. Fish and Shellfish Immunology, 3(4), 237-251.
Shin, G., Lee, H., Palaksha, K. J., Kim, Y., Lee, E., Shin, Y., Lee, E., Park, K., and Jung, T. (2006). Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf). Journal of Veterinary Sciences, 7(3), 293–295.
Thuvander, A., Fossum, C., and Lorenzen, N. (1990). Monoclonal antibodies to salmonid immunoglobulin: characterization and applicability in immunoassays. Developmental and Comparative Immunology, 14(4), 415-423.
Uchida, D., Hirose, H., Chang, P.K., Aranishi, F., Hirayabu, E., Mano, N., Mitsuya, T., Prayitno, S.B., and Natori, M. (2000). Characterization of Japanese eel immunoglobulin M and its level in serum. Comparative Biochemestry and Physiology B, 127, 525–532.
Uchuwittayakul, A., Rodkhum, C., and Srisapoome, P. (2025). Production of a monoclonal antibody specific to the IgM heavy chain of Asian seabass (Lates calcarifer Bloch, 1790) and its application in assessing health status following vaccination and challenges with Flavobacterium covae and Streptococcus iniae. Aquaculture, 594, https://doi.org/10.1016/j.aquaculture.2024.741445
Velázquez, J., Rodríguez, A., Aragón, H., Haidar, A., González, M., Valdés, R., Garay, H.E., Abreu, D.D., Ramos, Y., Cabrales, A., Morales, A., González, O., Herrera, F., Estrada, M.P., and Carpio Y. (2021). Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells. Fish and Shellfish Immunology, 110, 44-54.
Vesely, T., Reschova, S., Pokorova, D., Hulova, J., and Nevorankova, Z. (2006). Production of monoclonal antibodies against immunoglobulin heavy chain in common carp (Cyprinus carpio L.). Veterinární medicína, 51(5), 296-302.
Yang, S., Tang, X., Sheng, X., Xing, J., and Zhan, W. (2018). Development of monoclonal antibodies against IgM of sea bass (Lateolabrax japonicus) and analysis of phagocytosis by mIgM+ lymphocytes. Fish and Shellfish Immunology, 78, 372-382.
Zhang, W., Tang, X.Q., Sheng, X.Z., Xing, J., and Zhan, W.B. (2017). Development and application of monoclonal antibodies against IgM of black rockfish (Sebastes schlegeli). Journal of Fish Biology, 90(4), 1668-1675.