Production, Characteristic Analysis And Application Of Monoclonal Antibodies Against Vibrio Harveyi

Vibrio harveyi H06091was isolated from fugu rubripes dermal ulcer which cultivated inDalian fishponds. In the current, methods for detecting this kind of bacteria is time-consumingand complicated. The operation needs professionals to completed which is not conducive to thepromotion of field testing. Immunological detection methods mediated by monoclonal antibodyhas been widely used by its high specificity and high sensitivity.In this experiment, we used V. harveyi H06091inactivated by formaldehyde to immuneBalb/C mice. Using monoclonal antibody technology by cell fusion, screening and limitingdilution cloning,19monoclonal antibodies anti-V. harveyi H06091were prepared. Themonoclonal antibodies were named1B6,1D6,1A4,5B4,6C6,2E7,4A7,5B2,4E12,1G3,7D7,8E10,4D12,2B9,7D6,6E6,1G2,6G6and2C1respectively. The19monoclonal antibodieswere characteristic analyzed. Reference strains such as Vibrio splendidus, Vibrioparahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio damsela, Vibrio anguillarum,Shewanella spp., Fugu streptococcus, Aeromonas salmonicida, Edwardsiella tarda,2strains of V.harveyi and5other isolated strains of V. harveyi were used in the specificity analysis.Monoclonal antibodies4A7,4E1,7D7,8E10,4D12and2B9had have high specificity.Monoclonal antibodies2E7,5B2,1G2,2C1and1G3cross-reacted with some strains. Thespecificity of monoclonal antibodies1B6,1D6,1A4,5B4,6C6,7D6,6E6and6G6wererelatively poor. Subtype analysis were carried for monoclonal antibodies4A7,4E12,7D7,8E10,4D12,2B9,2E7,5B2,1G2and2C1. Monoclonal antibody7D7and8E10were IgGsubtype, while the remaining eight monoclonal antibodies were IgM subtype. Titer andsensitivity analysis were conducted for monoclonal antibodies4A7,4E12,7D7,8E10,4D12,2B9,2E7,5B2,1G2and2C1. The titers were between1:4to1:1024. The sensitivity ofmonoclonal antibody to detect V. harveyi can limit to5×105cells/mL. All monoclonal antibodieshad have strong stability.Monoclonal antibody8E10were selected to establish a double-antibody sandwichELISA method for rapid detection of V. Harveyi. The best concentration of polyclonal antibodywas1:12800determined by checkerboard titration, while the best concentration of monoclonalantibody was1:128. The dilution of goat anti-mouse IgG conjugated with alkaline phosphatase(AP) was1:2000. This detection method had high specificity which could detect V. harveyiH06091specially and had no cross rection with other bacteria strains. The sensitivity of thedetection method can reach5×106cells/mL. V. Harveyi is important pathogens in aquaculture which could cause disease outbreakamong a variety of aquatic animals causing serious economic losses. In our experiment, weprepared monoclonal antibody anti-V. Harveyi with high specificity and sensitivity. We alsoestablished a double-antibody sandwich ELISA method for rapid detection of V. Harveyi. Thematerial foundation was settled for the preparation of immunochromatographic test strip todetect V. Harveyi.