Abstract
Background: Accurate and easy-to-perform assays for the detection of antibiotic-resistant bacterial isolates producing AmpC β-lactamases are epidemiologically relevant, leading to more effective use of antibiotics and a comprehensive understanding of β-lactamase prevalence. We describe novel monoclonal antibodies (MAbs) against CMY β-lactamases and their application in immunoassays for the detection of CMY-producing bacterial isolates. Methods: Recombinant CMY-34 was expressed in Escherichia coli and used as an immunogen for MAb generation by hybridoma technology. Selected CMY-34-specific MAbs were comprehensively characterized by various immunoassays, computational analysis, and sequencing of their variable domains. To prove MAb reactivity with CMY β-lactamases, the antibodies were tested with CMY-producing bacterial isolates. For this purpose, the MAbs were applied in sandwich-type assays, such as sandwich enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and two-photon excitation (TPX) assay for immunodetection of CMY enzymes. Results: Two high-affinity MAbs raised against recombinant CMY-34 were characterized in detail. Both MAbs recognized CMY-34 β-lactamase in the Citrobacter portucalensis isolate. The analysis of MAb epitopes revealed their sequence homology among the members of the CMY family, suggesting their potential broad reactivity. Comprehensively characterized MAbs were successfully applied in sandwich ELISA and two rapid immunoassay formats that were tested with CMY-positive bacterial isolates. MAb-based immunoassays detected all analyzed CMY-positive isolates producing CMY-2, CMY-4, CMY-6, CMY-16, and CMY-34 β-lactamases. Conclusion: Novel MAbs raised against CMY-34 recognize common epitopes of CMY β-lactamases and can be applied for immunodetection of CMY β-lactamases in bacterial isolates.