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Development and characterization of monoclonal antibody against Vibrio harveyi lipopolysaccharide

HAO Gui-jie,SHEN Jin-yu,PAN Xiao-yi,CAO Zheng,YIN Wen-lin (Zhejiang Institute of Freshwater Fisheries,Huzhou 313001,China)  
The 8-week-old female BALB/c mice were immunized with a pathogenic V.harveyi GYC1108-1,which was isolated from diseased great yellow croaker in Zhejiang Province in 2003.Spleen cells collected from immunized mice after the fifth immunization were fused with SP2/0-Ag-14 myeloma cells.Indirect Enzyme-Linked Immunosorbent Assay(ELISA) was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones.After three cycles of subcloning,a McAb against the lipopolysaccharide(LPS) of GYC1108-1 was obtained,and was designated as 2F9.The obtained McAb 2F9 was proved to have high ELISA titers.The specificity test suggested that 2F9 reacted with V.harveyi,V.alginolyticus,V.parahaemolyticus,but did not react with V.mimicus,V.anguillarum,Aeromonas hydrophila and other isolates among 14 bacteria species tested.2F9 could recognize GYC1108-1 LPS,but the reaction was reduced after LPS oxidation by mild periodate acid.Immunoblotting after SDS-PAGE shows that 2F9 is specific for lipoid A of GYC1108-1.The LPS released into cultured medium in 48 hours was not more than 39 μg/mL semiquantitated by inhibition ELISA,which is by far less than the amount necessary for fish lethality.
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