| Objective:Gout is a kind of inflammatory arthritis caused by crystallization of monosodium urate(MSU).In recent years,with the change of dietary structure and other environment factors,the incidence rate of gout has gradually increased.Gout has become the diseases that seriously endangers human health.Allopurinol is used to treat gouty joints since 1963,and it has become the first-line clinical drugs for many years.However,serious adverse reactions have been found in clinical application.Studies have shown that allopurinol allergic reaction closely related with HLA-B*5801 gene.Therefore,genotype testing Provides a instruction for individualized medication and reduces adverse drug reactions.In this study,we aim to produce a monoclonal antibody targeting HLA-B*5801 peptides,and detect its specificity and sensitivity,We also discussed the possibility of the monoclonal antibody to used as a detecting antibody for the HLA-B*5801.Method:Hybridoma and subclones techniques were used to generate the mAb through immunized with the HLA-B*5801 polypeptide.Indirect ELISA assay and western blotting were applied to identify the mAb.Ion exchange chromatography purifies the monoclonal antibodies.The value of the monoclonal antibody was detected by double dilution method,IsoStrip Mouse Monoclonal Antibody Isotyping Kit Detects the subclass of the antibodies,ELISA and Western blot were employed to verify its specificity.Results:Two hybridoma cell line was obtained by monoclonal and subclonal screening.The two monoclonal antibody,named as 1E1 and 2A3 were produced by injecting the hybridoma cell into mice abdominal cavity,The subclass of monoclonal antibody was identified as IgGl and kappa,The antibody titer was 1014 and 1010,ELISA and Western blot were used to verify its specificity to HLA-B*5801 peptides.Conclusion:Our data suggests the monoclonal antibody we obtained can specific bind with HLA-B*5801 peptide,and the antibody can be used as detecting antibody for allopurinol allergy patients. |
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