Development of Competitive ELISA and CLEIA for Quantitative Analysis of Polymyxin B Long Xu 1 & Maksim Burkin 2 & Sergei Eremin 3 & Alberto C. P. Dias 1 & Xiaoying Zhang 1,4 Received: 4 November 2018 /Accepted: 13 February 2019 /Published online: 25 February 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 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 (IC 50 ) 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 77.4~106.1% (ic-ELISA) and 84.1~107.1% (ic-CLEIA), respectively. Keywords Polymyxin B (PMB) . Animal-derived food . Enzyme-linked immunosorbent assay (ELISA) . Chemiluminescent enzyme immunoassay (CLEIA) Introduction Polymyxins (PM) are pentacationic polypeptides with a com- mon structure: a cyclic heptapeptide, a linear tripeptide por- tion, and a fatty acyl (FA) tail which is linked to the N- terminus of the tripeptide part (Storm et al. 1977). They have five different agents, which are described and named as poly- myxins A–E (Kadar et al. 2013). They differ from each other in their amino acid sequences and FA chains. Among these molecules, only PMB and PME are available for clinical use (Cai et al. 2015). They are isolated from cultures of various strains of Bacillus polymyxa and related species (Liu et al. 2017; Pendela and Adams 2004). PMB has been used for the treatment of infections caused by gram-negative bacteria, especially the Pseudomonas aeruginosa and Escherichia coli (Gales et al. 2001; Orwa et al. 2000). It is the drug of first choice in the treatment of infections of the urinary tract, eye, meninges, and bloodstream. Many countries have promulgat- ed standards for residue limits of PME. The EU and China both set up PME residue limits of 150 μg/kg in the animal muscle, fat, and liver and 200 μg/kg in the kidney. However, there is no maximum residue limit for PMB in animal-derived foods and animal feed internationally. PMB has been known to show antiendotoxin activities, and it is reported to bind to endotoxin in vitro to suppress many of its activities (Guo et al. 2007; Ronco and Klein 2014). Owing to the emergence of multidrug-resistant (MDR) gram-negative antibiotics and the reduction of newly developed antibiotics, PMB has been in- creasingly used as a last-resort drug in the treatment of MDR gram-negative bacterial infections (Bergen et al. 2012a; Velkov et al. 2013). With the increasing use of PM in humans and animals, bacterial resistance has been on the increase (Li et al. 2018; * Xiaoying Zhang zhang@bio.uminho.pt; zhang.xy@nwsuaf.edu.cn 1 Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 2 Department of Immunology, I. Mechnikov Research Institute of Vaccines and Sera, Moscow, Russia 105064 3 Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia 119991 4 College of Veterinary Medicine, Northwest Agriculture and Forestry University, PostBox 19, Xinong Rd. 22, Yangling 712100, Shaanxi Province, China Food Analytical Methods (2019) 12:1412–1419 https://doi.org/10.1007/s12161-019-01477-9