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Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens associated with nosocomial and community infections that are difficult to treat owing to its resistance to all β-lactams and other classes of antibiotics. Reports of MRSA demonstrate the pathogen relevance and urgency for developing innovative diagnostic and treatment strategies against this microorganism. In this context, monoclonal antibodies (mAbs) represent a powerful tool for such purposes. Beta-lactam resistance in MRSA is caused by penicillin-binding protein 2a (PBP2a). The characteristics of PBP2a make this protein a potential target for immunobiologicals to combat this pathogen. This study describes the development of a recombinant Fab fragment from a mAb directed against the PBP2a protein, designed to identify and treat MRSA infections. The Fd and light chain coding sequences for Fab expression were amplified and ligated into the mammalian cell expression vector. Recombinant DNA constructs were used to transfect Expi293F cells expressing anti-PBP2a Fab. A purification based on ion-exchange chromatography was used for Fab separation, followed by analysis of antigen target recognition and interaction, either with the isolated antigen or with the antigen on the MRSA cell surface. The experimental approach allowed us to obtain significant Fab expression levels in the Expi293F system when transfecting the cells with the genetic constructs developed in pCDNA3.4 vector. Antigen target interaction assays revealed the capacity of Fab to recognize and interact with the PBP2a protein. Biodistribution analysis indicated serum Fab presence, in the serum, kidneys, lungs, and spleen, and a plasma half-life averaging 6-8 h., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)