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The production of the recombinant S monoclonal antibody involves the following steps:
1. Isolation of mouse scFv: Mice are immunized with human SARS-CoV-2 S (16-685aa) to stimulate an immune response. Splenocytes are obtained and RNA is isolated, followed by reverse transcription to obtain cDNA.
2. Generation of scFv: The variable regions of the mouse antibody's heavy and light chains are amplified from the cDNA using PCR. These regions are then combined to construct the scFv.
3. Cloning of scFv into an expression vector: The DNA sequence encoding the scFv is inserted into an expression vector. The vector contains the DNA sequence encoding the human IgG1 Fc region downstream of the scFv. Additionally, a DNA sequence encoding Biotin is introduced downstream of the Fc region to create the scFv-Fc-Biotin fusion construct.
4. Transfection and expression: The recombinant expression vector is transfected into a host cell line to enable the expression of the scFv-Fc-Biotin fusion protein.
5. Antibody purification: The cell culture supernatant, containing the recombinant S monoclonal antibody, is collected. The antibody is then purified using affinity chromatography.
6. Characterization and validation: The binding specificity of the recombinant S monoclonal antibody, conjugated with Biotin, is confirmed by testing its ability to recognize the human SARS-CoV-2 S protein using ELISA.
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