Preparation of monoclonal antibodies against SARS coronavirus nucleocapsid protein and preliminary analysis of antigenic epitopes

Objective:To prepare monoclonal antibodies with high specificity against different regions of N protein and to analyze antigenic epitopes of SARS-CoV N protein.Methods: Balb/ c mice were immunized with purified different region N protein to prepare McAb by hybridoma technique and select by indirect ELISA.Both of these McAbs were characterized for their antibody titre in the culture supernatant as well as ascites,class,subclass and affinity analysis.The antigen specificity for McAb were evaluated and confirmed by Western blot.The binding site and capability of McAb were observed by ELISA additive assay.Results: Seven hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N1 and two hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N2 were obtained.About the immunoglobulin subclass of McAb,six were IgG1,two were IgG2b and one was IgG3.Western blot showed that the McAbs reacted specifically with SARS-CoV N protein.ELISA additive assay indicated that two antibodies against SARS-CoV N1 recognized same epitopes,while others recognized distinct epitopes.Conclusion: We successfully obtained nine specific McAbs against the different region SARS-CoV N protein after expression and purification of the different part SARS-CoV N protein.It shows that the N1 McAb recognizs the N-terminus of the N protein whereas the N2 McAb recognizs the C-terminus.