Penicillin Binding Proteins-Based Immunoassay for the Selective and Quantitative Determination of Beta-Lactam Antibiotics

Abstract

An immunoassay method based on penicillin-binding protein (PBP) was developed for the quantitative determination of 10 kinds of beta-lactam antibiotics (BLAs). First, two kinds of PBPs, which are named PBP1a and PBP2x, were expressed and purified, and they were characterized by SDS-PAGE and Western-Blotting analysis. Then, the binding activity of PBP1a and PBP2x to template BLAs, cefquinome (CEFQ), and ampicillin (AMP) was determined by using AMP-horseradish peroxidase as the competitor. The effect of the buffer solution system, e.g., pH, ion concentration, and organic solvent, on the immune interaction efficiency between PBPs and BLAs was also evaluated. In the end, the based immunoassay method was developed and validated for the detection of 10 kinds of BLAs. Under optimal conditions, PBPs exhibited high binding affinity to BLAs. In addition, this method showed a high sensitivity for the detection of 10 kinds of BLAs with the limits of detection from 0.21 to 9.12 ng mL–1, which are much lower than the limits of the maximum residual of EU (4-100 ng mL–1). In the end, the developed PBP-immunoassay was employed for BLA detection from milk samples, and satisfactory recoveries (68.9–101.3%) were obtained.

Keywords: Penicillin-binding protein, Beta-lactam antibiotics, Immunoassay