Preparation and identification of monoclonal antibodies against structural proteins of SARS-CoV-2
Journal Title: Journal of Air Force Medical University - Year 2023, Vol 44, Issue 7
Abstract
Objective To prepare murine-derived monoclonal antibodies ( mAbs) against the S1 subunit of spike protein (S protein) and nucleoprotein (N protein) of severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2) for preliminary identification of their functions and applications, and to prepare human-mouse chimeric antibodies with high affinity and neutralizing activity. Methods BALB / c mice were immunized with the S1 subunit of S protein and N protein of the recombinant SARS-CoV-2, respectively. MAbs against the S1 subunit of S protein and N protein were prepared by conventional B-cell hybridoma technique, and their application values were evaluated by Western blotting and immunohistochemical staining. A sandwich ELISA method for detecting the soluble S protein and N protein was established. The neutralizing activity of specific antibodies was detected based on pseudovirus-luciferase detection system. Hybridoma cell lines secreting neutralizing mAbs against the S1 subunit of S protein were sequenced to prepare human-mouse chimeric antibodies. Results A total of 53 hybridoma cell lines secreting murine mAbs against the S1 subunit of S protein of SARS-CoV-2 were obtained, with clone numbers named XA326. 1 - XA326. 53, and 42 hybridoma cell lines secreting murine mAbs against SARS-CoV-2 N protein were obtained, with clone numbers named XA327. 1 - XA327. 42. Preliminary identification showed that 9 strains of mAbs against the soluble S1 subunit of S protein and 8 strains of mAbs against soluble N protein of SARS-CoV-2 could be used for Western blotting, while 9 strains of mAbs against the S1 subunit of S protein and 4 strains of mAbs against N protein of SARS-CoV-2 could be applied to immunohistochemical staining. A sandwich ELISA kit with mAb XA326. 7 as coating antibody and biotin-labeled mAb XA326. 19 as detection antibody was established to detect the soluble S1 subunit of S protein of SARS-CoV-2 in serum, and a sandwich ELISA kit with mAb XA327. 37 as coating antibody and biotin-labeled mAb XA327. 25 as detection antibody was established to detect the soluble N protein of SARS-CoV-2 in serum. XA326. 4, XA326. 7, XA326. 8, and XA326. 49 mAbs had neutralizing activity, which could block viral infection of human ACE2-positive cells in the pseudovirus infection experiment. Conclusion The preparation of murine mAbs against the S1 subunit of S protein and N protein of SARS-CoV-2 can be used in a variety of immunological detection techniques. The neutralizing antibodies XA326. 4, XA326. 7, XA326. 8, and XA326. 49 have potential clinical application value.
Authors and Affiliations
WANG Yuling, JIN Boquan, LI Na, TIAN Ying, DONG Yun, LI Qi, MA Ying, ZHUANG Ran
Keywords
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