А Method for the Quantitative Determination of the Active Receptor of Beta-lactam Antibiotics BlaR-CTD for Bioanalytical Applications

Abstract

A sandwich bioassay for the quantitative determination of the recombinant beta-lactam receptor BlaR-CTD possessing ligand binding activity and immunoreactivity has been developed. In the bioassay system, BlaR-CTD present in a biological liquid or standard sample binds via its receptor site to ampicillin immobilized in a microplate well and interacts through the epitopes of its peripheral structure with specific polyclonal antibodies. The analytical sensitivity of the method proved to be 2 ng/mL, and its concentration range was 5–215 ng/mL. In the processes of heterological expression, isolation and reagent forms preparation, the biological activity of BlaR-CTD was monitored and its stability was evaluated. High purity recombinant beta-lactam receptor BlaR-CTD was obtained. The protein was shown to have a sufficiently high resistance to denaturation by chaotropic agents (urea and guanidine hydrochloride), and it was stable over a wide pH range. Also, we proposed the constructions and procedures of competitive bioassays for beta-lactam antibiotics using microplates (analytical sensitivity – 0.02 ng/mL, IC50 = 0.28 ng/mL) or chromatographic test-strips (detection limit 1–2 ng/mL), which are based on the receptor and antigenic properties of BlaR-CTD.