Preparation Of Monoclonal Antibodies And Development Of Rapid Detection Methods For Vibrio Harveyi

Vibrio harveyi is an opportunistic pathogen in seawater and can cause severe vibriosis among aquaculture species and lead to severe economic losses.As the traditional enrichment culture and molecular detection method are complex and time-consuming,they can not be applied in grassroots units.Therefore,there is an urgent need to develop a rapid and simple detection method for V.harveyi on-site detection.In this study,to prepare monoclonal antibodies(MAbs)against V harveyi,BALB/c mice were immunized with formalin-killed V harveyi,and then spleen lymphocytes were fused with Sp2/0 by PEG solution or by cell electrofusion.Two stable hybridoma cell lines 4D9 and 2B9 were obtained following the subcloning procedure,and then injected intraperitoneally into the mice for ascite production.After 7 to 10 days,ascite was collected and MAb in ascite fluid was purified by a protein G agarose column,and then MAb was labeled by colloidal gold to prepare test strip for V.harveyi.The isotype of the two MAbs were analized.The results displayed both of the MAbs were IgG1,which suggested that they can not be applied in double antibodies sandwich test strip simultaneously.Therefore,PAb and MAb were chosen to prepare test strip.Since the limit of detection(LOD)for V harveyi was as high as 108 CFU/ml,the test strip can not be applied in practical detection.An ECL-based dot blot assay was developed for the rapid and sensitive detection of V.harveyi based on MAbs against V.harveyi.The blocking buffer,membrane and incubation time of the dot blot assay were optimized,and enhanced chemiluminescent(ECL)substrate replaced the traditional TMB substrate.According to the optimization results,the LOD for V.harveyi was 2×105 CFU/ml(103 CFU/spot)for pure bacterial suspension sample.Compared with the traditional dot blot assay,the incubation time was shortened from 8 h to 2 h.The LOD was 50-fold more sensitive than the traditional dot blot assay(1×107 CFU/ml).Furthermore,when compared with indirect ELISA,the dot blot assay showed approximately 1000-fold higher sensitivity(CFU/CFU).After the test sample was pre-enriched in turbot homogenates for 6 h before the dot blot analysis,the LOD for V.harveyi was 10 CFU/ml.ECL-based dot blot was not affected by complex background and can be operated easily,so it can be applied in grassroots units.