Abstract
Increasing attention is now being directed to the utilization of polymyxin B (PMB) as a last-line treatment for life-threatening infections caused by multidrug resistant Gram-negative bacteria. Unfortunately, polymyxins resistance is also increasingly reported, leaving a serious threat to human health. Therefore, the establishment of rapid detection methods for PMB residues is highly essential to ensure public health. In this study, two monoclonal antibodies (mAb; 2A2 and 3C6) were obtained using PMB-bovine serum albumin as the immunogen and PMB-ovalbumin as the coating antigen, which were prepared with N-(γ-maleimidobutyryloxy) succinimide ester and glutaraldehyde as cross-linking agents, respectively. Through an indirect competitive enzyme-linked immunosorbent assay, resultant two mAbs were compared and the results indicated that 3C6 showed higher sensitivity with a half maximum inhibition concentration of 13.13 ng/mL. Based on 3C6, a gold nanoparticles (AuNPs)-based immunochromatographic test (ICT) strip was then established, the mechanism of which is that free PMB competes with the fixed coating antigen to combine with mAb labeled by AuNPs. Using ICT strip to detect milk and animal feed samples revealed the visible detection limits were 25 ng/mL and 500 μg/kg, respectively and the cutoff limits were 100 ng/mL and 1000 μg/kg, respectively. The ICT strip provides results within 15 min, facilitating rapid and semi-quantitative analysis of PMB residues in milk and animal feed.