Objective To prepare monoclonal antibody (McAb) against COVID-19 in mice by using mRNA-LNP, the receptor binding region of the S protein of COVID-19 (RBD), as the immunogen. Methods This study used mRNA LNP containing the RBD protein sequence as an immunogen, . Immunization of Balb/c mice with RBD mRNA LNP via intramuscular injection. The RBD antibody titer in the serum of the mice was detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA) ; After achieving the standard for serum titer detection, stable hybridoma cells secreting RBD monoclonal antibodies were constructed using hybridoma fusion cell technology, followed by ascites production and antibody purification; Use immunofluorescence (IF), flow cytometry (FCM), enzyme-linked immunosorbent assay (ELISA), Western blot and other methods to identify its biological activity and detect the specificity of the obtained antibodies. Result After re screening, two hybridoma cells capable of stably secreting RBD McAbs were obtained and named 2D7D11 and 9B6H5, respectively. After testing, the Ig subclasses of 2D7D11 and 9B6H5 are both IgG2b, with the light chain being the kappa chain; Through IF, FCM, ELISA, and Western blot analysis, it was confirmed that the 2D7D11 and 9B6H5 RBD McAbs exhibit high specificity towards RBD protein or S-RBD (e1) - PET28 recombinant protein expressed in RBD mRNA LNP transfected cells. Conclusion This study successfully prepared two anti RBD specific monoclonal antibodies, 2D7D11 and 9B6H5, This provides an experimental basis for the delivery, translation, and modified expression of mRNA LNP as a novel immunogen in animals, the autonomous generation of humoral immune responses to produce antibodies, and the development of related detection methods and functional exploration; This provides an effective novel immune strategy for antibody development projects that are hindered by the inability to prepare effective immunogens due to various reasons.
Key words
receptor binding region of the S protein of COVID-19 (RBD) /
mRNA-LNP /
immunogen /
immune strategy /
monoclonal antibody
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