Development of Competitive ELISA and CLEIA for Quantitative Analysis of Polymyxin B

Abstract

Polymyxin B (PMB), a member of polypeptide antibiotics, is widely used for the treatment of infection in animals such as cattle, sheep, pigs, and chickens. However, it is toxic on the kidneys and nervous system, and polymyxin resistance is increasingly reported, which leaves a serious threat to human health. Therefore, it is essential to establish rapid methods for detecting PMB with high sensitivity and specificity. In this study, an anti-PMB polyclonal antibody (pAb) was obtained by immunizing New Zealand white rabbits with PMB conjugated with glycosylated bovine serum albumin (GBSA). Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) were developed. Under the optimal conditions, inhibitory concentrations (IC50) of PMB were 257.1 ng/mL (ic-ELISA) and 250.8 ng/mL (ic-CLEIA); the limits of detection (LOD) were 17.4 ng/mL (ic-ELISA) and 14.5 ng/mL (ic-CLEIA), respectively. Cross-reactivity of the pAb toward polymyxin E (PME) was 257.1%, and no response was found with other antibiotics. The recovery rates in spiked meat samples were 77.4 similar to 106.1% (ic-ELISA) and 84.1 similar to 107.1% (ic-CLEIA), respectively.