RESEARCH PAPER Mixed immunoassay design for multiple chemical residues detection Yanshen Li & Peng Li & Xiangshu Luo & Zhihui Hao & Zhanhui Wang & Jianzhong Shen & Xingyuan Cao & Suxia Zhang Received: 21 November 2012 / Revised: 16 January 2013 / Accepted: 22 January 2013 / Published online: 5 February 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horse- radish peroxidase to deoxynivalenol (DON) and orbifloxa- cin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23 ∼ 68 ng mL -1 for DONs and 4.1 ∼ 49 ngmL -1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng mL -1 for DONs and 0.59–6.9 ngmL -1 for QNs, which was lower than the maximum residue levels. Recoveries in neg- ative samples spiked at concentrations of 100, 200, and 500 ngmL -1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants. Keywords Deoxynivalenol . Quinolone . Analysis . Integrated enzyme-linked immunosorbent assay Introduction Deoxynivalenols (DONs; Fig. 1), type B of the trichothe- cenes, are mainly produced by Fusarium culmorum. DON and its main metabolites, deepoxy-deoxynivalenol (DOM- 1) and 3-acetyl-deoxynivalenol (3-AcDON), were first iso- lated from barley in Japan, and now they are agriculturally important mycotoxins relevant to human and animal health [1–3]. Quinolones (QNs; Fig. 1), which show broad- spectrum antimicrobial activity, are often used for the pre- vention and the treatment of infectious diseases caused by pathogenic bacteria [4, 5]. Recently, numerous studies concerned on the toxicity of these compounds have been reported [2, 3, 6]. In order to keep consumers away from these potential harmful compounds, the maximum levels for DONs [7] and maximum residue levels (MRLs) for QNs [8] have been settled in different regions. To deal with these questions, sufficiently sensitive ana- lytical methods are needed in order to implement these policies. Traditionally, several analytical methods were de- veloped in the past decades, including high-performance liquid chromatography (HPLC) [9, 10], liquid chromatogra- phy tandem mass spectrometry (LC-MS/MS) [11], gas chro- matography (GC) [12, 13], thin-layer chromatography [14], and enzyme-linked immunosorbent assay (ELISA) [15, 16]. Chromatographic techniques, such as HPLC and GC, are sensitive and selective. However, they are time-consuming and expensive. In consequence, immunoassays are gradual- ly employed for its simplicity, low cost, and high throughput advantages. For multi-residue analysis, scientists made lots of efforts to prepare an antibody which could recognize a group of compounds. In addition, a new technique of recombinant Yanshen Li and Peng Li contributed equally to this work. Y. Li : P. Li : X. Luo : Z. Wang : J. Shen : X. Cao : S. Zhang (*) Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 1000193, China e-mail: suxia@cau.edu.cn Z. Hao Agricultural Biological Pharmaceutical Laboratory, College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China Anal Bioanal Chem (2013) 405:3307–3312 DOI 10.1007/s00216-013-6780-x