Screening of mouse-derived monoclonal antibodies against the receptor binding domain of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein

Abstract

Objective To prepare and screen out monoclonal antibodies against the receptor binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV)spike (S) protein in mice. Methods The RBD of MERS-CoV S protein expressed in the insect-baculovirus system was purified and then used to immunize the female BALB/c mice. The spleen cells collected from the mice were fused with myeloma Sp2/0 cells. The positive hybridoma cells were obtained by using limited dilution method. Enzyme-linked immunosorbent assay (ELISA), Western blot assay and neutralization test based on the MERS-CoV pseudovirus were performed for further screening and identification. Results Twelve strains of hybridoma cells that produced the monoclonal antibodies against RBD of MERS-CoV S protein were screened out. All of the 12 monoclonal antibodies (McAbs) could have specific reaction with the RBD of MERS-CoV S protein as indicated by the results of ELISA. Of the 12 McAbs, two were identified as the immunoglobulin M (IgM) isotype and the rest were IgG1 isotype by using double antibodies sandwich ELISA. Four McAbs including 1F1, 2E4, 3C3 and 3E6 were identified as having neutralizing activity by the neutralization test based on MERS-CoV pseudovirus. Results of the Western blot assay showed that the four McAbs (1F1, 2E4, 3C3 and 3E6) could have specific reaction with the RBD of MERS-CoV S protein, but no cross-reaction with that of SARS-CoV S protein. Conclusion Twelve mouse-derived McAbs against the RBD of MERS-CoV S protein were obtained. The prepared hybridoma cells showed the characteristics of high specificity and stability in antibody secretion. Four out of the 12 McAbs were proved to have neutralizing activity. Key words: Middle East respiratory syndrome; Coronavirus; Spike protein; Receptor binding domain; Monoclonal antibody