Analytical Methods Hapten synthesis, monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk Zhanhui Wang a , Tiejun Mi a , Ross C. Beier b , Huiyan Zhang a , Yajie Sheng a , Weimin Shi a , Suxia Zhang a , Jianzhong Shen a,⇑ a College of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory For Food Quality and Safety, Beijing 100193, People’s Republic of China b Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, TX 77845-4988, USA article info Article history: Received 25 April 2013 Received in revised form 13 August 2014 Accepted 23 August 2014 Available online 6 September 2014 Keywords: Cross-reactivity Enzyme-linked immunosorbent assay Erythromycin Hapten incorporation Macrolides Monoclonal antibody abstract Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, preven- tion and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or oval- bumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC 50 values for erythromycin of 14.40 and 0.94 lgL 1 , respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Erythromycin is one of the most commonly used macrolide antibiotics in veterinary medicine to treat respiratory diseases and enteric infections in swine, cattle, sheep and poultry. It has also been used on a large scale as a feed additive and also delivered via drinking water for animal growth promotion (Díaz-Cruz & Barceló, 2007; McGlinchey, Rafter, Regan, & McMahon, 2008). In humans, erythromycin is often administered to those who are allergic to penicillin, and it has proven to be a safe and effective therapy for a number of commonly encountered infections (Ray et al., 2004). In aquaculture, erythromycin is used to treat infec- tions from Gram-positive bacteria, such as Lactococcus garvieae in trout (Lucchetti et al., 2005). Erythromycin has low toxicity, but residues in food animals might provoke allergic reactions in some hypersensitive individuals or lead to drug-resistant pathogenic bacteria (Thong, 2010; Wieczorek, Kania, & Osek, 2013). In addition, the use of these antibiotics has resulted in their release into the environment through different pathways; thereby, posing a potential risk to the ecosystem, as well as human and animal health by shifting the physiological profile of microbial communi- ties (Koike et al., 2007; Maul, Schuler, Belden, Whiles, & Lydy, 2006). For these reasons, the European Union (EU), Ministry of Agriculture (MOA) in China and other international bodies have established maximum residue limits (MRLs) for erythromycin in food-producing animal species (Font et al., 2008). For example, the EU and MOA in China have set MRLs for erythromycin in milk at 40 lgL 1 . Currently, methods for the determination of erythromycin in different matrices are often based on chromatographic techniques using various detection systems (Avramov Ivic ´ et al., 2008; Hu et al., 2010; Minh, Lam, & Giao, 2011; Tao et al., 2012; Ye, Weinberg, & Meyer, 2007). Although these methods are sensitive and selective, there is still a need for rapid and cost-effective alter- natives to screen large numbers of samples, and in particular for on-site detection. An effective alternative, based on specific anti- gen–antibody interactions are immunoassays, which are low cost http://dx.doi.org/10.1016/j.foodchem.2014.08.104 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +86 10 6273 2803; fax: +86 10 6273 1032. E-mail address: sjz@cau.edu.cn (J. Shen). Food Chemistry 171 (2015) 98–107 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem