Objective Preparation of monoclonal antibody against monkeypox virus(MPXV)A35R protein and a related double antibody sandwich ELISA assay was established and validated.Methods The monkeypox virus A35R protein was expressed using prokaryotic expression system and immunized BALB/c mice,the anti-A35R monoclonal antibody was pre-pared by hybridoma technique,the specificity and binding activity of the monoclonal antibody were charaterized;a double-antibody sandwich ELISA assay was established,and its linear range,detection limit,specificity,accuracy and precision were verified.Results Seven monoclonal antibodies against monkeypox virus A35R were prepared.Western blot analysis dem-onstrated that all seven monoclonal antibody strains exhibited specific reactivity with the MPXV A35R protein and inactiva-ted MPXV.The monoclonal antibody binding activity of the seven strains were from 11.35 ng/mL to 27.73 ng/mL.After screening for antibody pairs,7G5 and 2F5 were identified as the optimal antibody pair for the double-antibody sandwich ELISA method.The minimum detection limits for inactivated MPXV antigen were 1.250×104 TCID50/mL and 1.95 ng/mL for MPXV A35R protein,the linear range was 3.91-125.00 ng/mL,the linear regression equation was y=1.732 0x-0.920 8,R2=0.984 3;the method is highly specific and does not cross-react with other viruses and proteins;the accuracy validation results showed that the recovery of viral antigens was between 96.94%-110.04%;inter-batch CV ranged from 0.26%to 8.13%,and intra-batch CV ranged from 2.10%to 5.48%.Conclusion An anti-monkeypox virus A35R protein monoclonal antibody was successfully prepared,and a related double-antibody sand-wich ELISA method was established and fully validated,the method can be employed for the purpose of quality control in the production of subunit monkeypox vaccines based on the A35R protein.
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