Development and characterization of anti-recombinant PBP2a antibodies for preliminary detection of methicillin-resistant Staphylococcus aureus

Abstract

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health and veterinary concern due to the widespread dissemination of the mecA gene, which encodes penicillin-binding protein 2a (PBP2a). Although PBP2a-based diagnostic assays are widely used, antibodies developed from locally derived MRSA strains remain limited despite regional genetic variation. Aim: This study aimed to develop and characterize anti-recombinant PBP2a (anti-rPBP2a) antibodies using recombinant mecA-derived proteins from clinical MRSA isolates in Indonesia and assess their preliminary applicability for MRSA detection using immunoblotting and latex agglutination assays. Methods: Two mecA gene fragments corresponding to amino acid positions 341–453 and 341–670 were amplified from Indonesian MRSA isolates, cloned into pET-24a(+), expressed in Escherichia coli BL21 (DE3), and purified to obtain recombinant PBP2a (rPBP2a) antigens. BALB/c mice were immunized with soluble and insoluble antigen preparations. Antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA), and antibody reactivity was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, dot blot assays, and latex agglutination against MRSA and methicillin-susceptible Staphylococcus aureus (MSSA) isolates. Results: Both recombinant constructs were successfully expressed, yielding proteins of approximately 14 and 40 kDa for the mecA341–453 and mecA341–670 constructs, respectively. The Elution B (EB) antigen fraction induced the strongest immune response, with ELISA optical density values of 2.4–3.5 during booster immunizations and ascites titers of 3.41–3.50. Western blotting revealed a distinct immunoreactive band at approximately 40 kDa, consistent with the predicted molecular weight of rPBP2a. Anti-rPBP2a antibodies generated against the EB antigen fraction showed stronger and more consistent reactivity than those raised against Pellet A and Pellet B antigens in dot-blot assays. In latex agglutination assays, EB-derived antibodies produced visible agglutination with MRSA isolates but not with MSSA, supporting their preliminary applicability for differentiating MRSA from MSSA. Conclusions: To the best of our knowledge, this study is the first to develop and characterize anti-rPBP2a antibodies derived from Indonesian MRSA isolates. These antibodies demonstrated strong immunoreactivity and consistent recognition of rPBP2a across multiple assay formats, providing a promising foundation for the future development of rapid and affordable MRSA detection methods in veterinary and clinical settings.