Purification Of Cholera Toxin And Preparation And Application Of Monoclonal Antibodies Against Cholera Toxin

State Key Laboratory for Infectious Disease Prevention and Control National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and PreventionObjectivesThe aim of this study was to establish an effective way to purification of CT protein from Vibrio cholerae culture supernatant and preparation of hybridoma cell lines anti-CT MAbs.Heat-labile toxin was cloned,expressed and purified in order to evaluate the specificity of anti-CT MAbs.BA-ELISA was also developed for CT determination and this method provides a technique reference for Vibrio cholerae toxigenic strains diagnosis.MethodsVibrio cholerae strain 569B was cultured in AKI medium.Crude cholera toxin was recovered from the culture supernatant by coprecipitation with sodium hexametaphosphate.Further purification was carried out by phosphocellulose column and immobilized D-galactose column chromatography successively.High concentration of CT was obtained by centrifugal ultrafiltration.The purity and activity of purified CT were judged by SDS-PAGE,Western Blot,and GM1-ELISA. Hyperimmune Balb/c mice were immunized with purified CT.Hybridoma cells lines of anti-CT MAbs were prepared by hybridoma technique,and then screened by indirect ELISA.The ascites were developed by injecting the hybridoma cells into abdominal cavity of Balb/c mice.The isotypes of the induced antibody were identified by Mouse Monoclonal Antibody Isotyping Reagents.Hitrap rProtein A FF purification column and euglobulin precipitation method were used for the purification of MAbs and PAbs,respectively.The LT gene was amplified by PCR from E.coli 10407 and inserted into pUC-19.The mutated gene was constructed and the sequences were analyzed.Then the gene encoding Heat-labile toxin(LT) was cloned into the expression vector pET-30a and transformed in E.coli BL21(DE3). GM1-ELISA was used as a method to screen LT expression strains and LT was purified from the supernatant of bacteria lysate by Immobilized D-galactose column. SDS-PAGE,MS,and Vero cell toxicity test were used as ways to verify the purity and normal biological character.Anti-CT MAbs were labeled with biotin.The optimal antibodies combination,antibody coating concentration,and biotinylated monoclonal antibody dilutions were determined.The limit value of CT assay was defined and different bacteria culture supernatant was assayed by BA-ELISA method.ResultsHigh purity and activity of CT(85 kDa) was obtained.Five hybridoma cell lines were developed,which could produce specific anti-CT MAbs.Isotypes of MAbs were identified as IgM.The MAbs could bind CT specifically by ELISA.High purity of anti-CT MAbs and PAb were obtained,and also the LT,which has high biological activity.The optimal antibodies combination was CTPAb as the coating antibody and CTMAb-XU-22 as the first antibody.The best coating concentration and biotinylated monoclonal antibody dilutions were 20μg/ml and 1:50,respectively.The limit value of CT assay was 2 ng.The result of bacteria culture supernatant assayed by BA-ELISA method was consistent with PCR method.Moreover,BA-ELISA could be used as a method to discriminate Vibrio cholerae toxigenic strains with non-toxigenic strains.ConclusionsAn effective way was established to purification of CT protein from culture supernatant and anti-CT MAbs were prepared successfully.BA-ELISA was successfully developed to detect CT.This method combined the high affinity of polyclonal antibodies(PAb),good specificity of monoclonal antibodies(MAb),and the amplification effect of BAS,which greatly improved the sensitivity and specificity and could be suitable for CT assay.This detection method may also provide a basis for the diagnosis of Vibrio cholerae toxigenic strains.