Synthetic single-domain antibodies to inhibit beta-lactamases

The invention includes 5 sdAbs (B2, B3, B4, B5, B6) with inhibitory activity against beta-lactamase from a broad range of gram-negative bacteria. These antibodies are capable of inhibiting beta-lactamase activity individually and exhibit synergistic properties when combined. The antibodies were obtained through serial selections from sdAb libraries displayed on the surface of yeast. Patent: P202230441. The original library contains approximately 10^9 sdAb combinations generated via PCR. After serial selection rounds, 200 sdAbs with high antigen-binding capacity were isolated, and those with the highest affinity for the antigen and strong inhibitory activity against beta-lactamase were selected. The sdAbs with the greatest binding capacity to the beta-lactamase antigen were chosen using an ELISA assay, in which 200 different antibodies were tested for their ability to bind to the selected antigen. The proteins with the highest antigen-binding capacity were individually produced in E. coli and purified by chromatography. The beta-lactamase inhibitory capacity was assessed through colorimetric nitrocefin assays. This type of assay provides quantitative data that can be used for enzymological studies. The isolated sdAbs have inhibition constants (Ki) ranging from 2.4 to 5.2 µM, meaning they can inhibit beta-lactamase enzymatic activity at very low concentrations.