Preparation and Application of Immunoglobulin Monoclonal Antibody from Largemouth Bass Micropterus salmoides
ZHANG Chengsai1, YIN Wenlin2, LI Ming3, CAO Zheng2, LIN Lingyun2, PAN Xiaoyi2, SHEN Jinyu2, WANG Chunfeng1, YAO Jiayun1,2, YANG Guilian1
1.Jilin Provincial Engineering Research Center of Animal Probiotics, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China; 2.Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China; 3.Jinhua Fisheries Technology Promotion Station, Jinhua 321051, China
Abstract：Serum immunoglobulin from largemouth bass Micropterus salmoides was purified by saturated ammonium sulfate and followed by affinity column chromatography extraction, then BALB/c mice were immunized with purified immunoglobulin, and the immunized spleen cells were fused with Sp2/0 mouse myeloma cells by using hybridoma-monoclonal antibody technology. Antibody titer, sensitivity and specificity of monoclonal antibodies were further determined by ELISA method. The formaldehyde-inactivated largemouth bass Largemouth bass ranavirus (LMBV) vaccine was prepared to immunize largemouth bass, and the antibody level after immunization was detected by the prepared monoclonal antibody. Two monoclonal antibody hybridoma cell line named A9E7and C9B9 were obtained, and ascites antibody titer of A9E7and C9B9 were reached up to 1∶2.187×106 and 1∶7.29×105, respectively. The measuring sensitivity of the A9E7 to purified IgM was as low as 10 ng, and the corresponding sensitivity of C9B9 was 20 ng. The results of cross reaction showed that both A9E7 and C9B9 binded specifically to the immunoglobulin of largemouth bass, A9E7 binded to the serum immunoglobulin of sea perch Lateolabrax maculatus, and C9B9 binded to mandarinfish Siniperca chuatsi. However, no cross reaction was found with grass carp Ctenopharyngodon idellus, crucian carp Carrassius auratus, loach Misgurnus anguillicaudatus, bluntnose black bream Megalobrama amblycephala,Acrossocheilus fasciatus, yellow catfish Pelteobagrus fulvidraco, topmouth culter Culter alburnus, ricefield Monopterus albus, silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis. After immunizing the LMBV, the antibody level in largemouth bass was increased significantly with peak at 35 d, and with antibody titer of 1∶1280. Monoclonal antibody against immunoglobulin of largemouth bass was prepared with strong specificity and high sensitivity, which can be used for early disease monitoring, immune response regularity and vaccine development of largemouth bass.
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2022, Vol. 41 
