Preparation Of Bispecific Monoclnal Antibody Against Recombinant VacA And HpaA Protein Of Helicobacter Pylori

Objective: The present study was intended to prepare a kind of the bispecific Monoclonal antibody(BsMcAb) against the protein of VacA and HpaA in Helicobacter pylori by the cell engineering method,to identify its biological,immunological,physicochemical characteristics. It would lay a foundation for the development of diagnoses and treatment for the H.pylori infection.Methods:The pQE30-V/H-DH5αwas extracted from the genetic engineering E.coli and identified by 12% SDS-PAGE.We take the purified protein as immunogen.The BALB/c mice were immunized with the recombinant VacA-HpaA fusion proteins of Helicobacter pylori for four times.The immune interval was two weeks. And 100～150μg the recombinant VacA-HpaA fusion proteins per mouse was intraperitoneally injected on three days before the hybridization.The spleen cells of the immunized mouse were hybridized with SP2/0 cells by 50%PEG(MW 4000).The hybridoma cells were selected with HAT culture medium. The antibody-secreting hybridoma cells were primary detected by the indirect ELISA using the purified rVacA as the antigen,and coloned by limited dilution to obtain the hybridoma cell lines which can stably secret the McAb against rVacA. The spleen cells of the immunized mouse were hybridized with the hybridoma cells obtained by 50%PEG(MW 4000).The hybri-hybridoma cells were selected with HAT culture medium. The antibody-secreting hybri-hybridoma cells were primary detected by the indirect ELISA using the purified rVacA and rHpaA as the antigen respectivly,and coloned by limited dilution to obtain the hybri-hybridoma cell lines which can stably secret the BsMcAb against rVacA and rHpaA.The chromosomes of hybri-hybridoma,SP2/0 and the spleen cells of the immunized mouse were counted. The stability of the BsMcAb-positive cell lines were detected by thirty subcultures in vitro,being frozen and thawed repeatedly for 6 times and thawed after frozen for 1.5 months.The ascites BsMcAb and the culture supernatant were prepared.The ascites BsMcAb was purified by the saturated ammonium sulfate and mmunoadsorption chromatography. Bradford measured their protein concentrations. the immunological characteristic of BsMcAb were analysed by indirect ELESA and Western blot. The Ig class of BsMcAb was identified by the Mouse McAb Isotyping Test Kit (10 assay, b.v). The antibody titers and antibody affinity constant of ascites BsMcAb were measured. The stability tests of ascites BSAb under different pH were performed. The stability test of the BsMcAb under different pH was as following:the ascites BsMcAb was diluted with the buffers of pH2.2,pH7.4 and pH9.6(the normal mouse serum as the negative control),then kept at 4℃for 24 hours and 48 hours respectively. The indirect ELISA was established with the purified bispecific monoclonal antibody.Results: The pQE30-V/H-DH5αwas abstracted from the genetic engineering E.coli and identified by 12%SDS-PAGE. The BALB/c mice were immunized successfully using antigen.Thirty-seven wells of antibody-secreting hybri-hybridoma cells against rVacA and rHpaA were primary obtained by twice cell hybridations,HAT selection and antibody detection. Three hybri-hybridoma cell lines can secret the BsMcAb against rVacA and rHpaA continuously after coloning.The BsMcAb can bind to the purified rVacA or rHpaA protein . The hybri-hybridoma cell lines was named fB8,fC5 and fE7,and one of them, the fB8 has the most satisfactory stability.The BsMcAb can be secreted by fB8 after thirty subcultures in vitro,being frozen and thawed repeatedly for 6 times and thawed after frozen for 1.5 months. The chromosomes of hybri-hybridoma,SP2/0 and the spleen cells of the immunized mouse were 127±5,64士3 and 40 respectively.The Ig class of fB8,fC5 and fE7 BsMcAb all were IgG1.The titers of the ascites BsMcAb-fB8 were 5.12×105. Its protein concentration was 11.32 mg/ml.ELISA and Western blot indicated that the antibodies were fairly specific for VacA and HpaA. The stability of the BsMcAb decreased under the acidity and alkalinity pH.Conclusions: Three hybri-hybridoma cell lines secreting bispecific monoclonal antibody against against rVacA and rHpaA in Helicobacter pylori established. BsMcAbs against rVacA and rHpaA were successfully obtained and purified,and showed good specificity and stability ,which might be used for prevention and cure of Helicobacter pylori and exploitation of test kit for Helicobacter pylori .and then play an important role in the study of H.pylori infection.