ABSTRACT
Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.