title: Production, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus).
abstract: Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD.
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Tang
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
authors.forename: Fuguo
authors.initials: F
authors.lastname: Liu
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
authors.forename: Xiuzhen
authors.initials: X
authors.lastname: Sheng
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xing
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No.1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, PR China. Electronic address: wbzhan@ouc.edu.cn.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Zhan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D029941:Fish Proteins / Q000276:immunology* / Q000378:metabolism; D005412:Flatfishes / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry / Q000662:veterinary; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D007075:Immunoglobulin M / Q000378:metabolism*; D007138:Immunoglobulin delta-Chains / Q000276:immunology* / Q000378:metabolism; D016131:Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D029941:Fish Proteins; D007075:Immunoglobulin M; D007138:Immunoglobulin delta-Chains
keywords: Flounder; Immunoglobulin D; Lymphocyte subset; Monoclonal antibody