Research paper Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues Hongyan Zhang a,b , Yan Zhang a , Shuo Wang a, ⁎ a Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300222, PR China b College of Life Science, Shandong Normal University, Jinan 250014, PR China article info abstract Article history: Received 15 February 2008 Received in revised form 18 April 2008 Accepted 22 April 2008 Available online 27 May 2008 Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip- stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2- horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1– 5 μg L - 1 or 1–10 μg L - 1 in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high- performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle(flow-through or dip-stick) or milk(only dip-stick). © 2008 Elsevier B.V. All rights reserved. Keywords: Sulfonamide Flow-through ELISA Dip-stick ELISA Strip assay 1. Introduction Sulfonamides share a common p-aminobenzoyl ring moiety with an aromatic amino group at the N4-position, differing in the substitution at the N1-position. Their back- bone and the structures of the sulfonamides described in this paper are shown in Fig. 1 . These synthetic compounds are widely used for therapeutic and prophylactic purposes in both human beings and animals, sometimes being used as additives in animal feed. Franco et al. (1990) and Mcevoy et al. (1999) have shown that if the proper withdrawal periods are not observed before slaughtering or milking of the medicated animals, meat and milk from these animals may be contaminated with residual sulfonamides. Furthermore, the main risk from abundant use of antimicrobials in animals is that bacteria may develop resistance to them. In European Union, Canada and the USA, the maximum residue limit (MRL) of total sulfonamides in edible tissues is 100 μg kg - 1 , and 20 μg kg - 1 in Japan. Huang et al. (2007) and Sun et al. (2007) have reported the instrumental analysis such as high-performance liquid chro- matograph (HPLC) which has been used successfully with high sensitivity and reliability for analysis of sulfonamides in different matrices. However, these methods require well- equipped laboratory, trained personnel, high capital expen- diture and involve time-consuming sample preparation steps. Sheth and Sporns (1991), Pastor-Navarro et al. (2007), Haas- noot et al. (2000), and Muldoon et al. (1999) have reported Journal of Immunological Methods 337 (2008) 1–6 Abbreviations: ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography; UV, ultraviolet–visible; MRL, maximum residue limit; LOD, the limit of detection; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; TMB, 3,3′,5,5′-tetramethylbenzidine; PBST, phosphate- buffered saline with 0.05% Tween 20; SIZ, sulfisozole; STZ, sulfathiazole; SME, sufameter; SPMX, sulfamethoxypyridazine; SP, sulfapyridine; SMT, sulfamethizole; SCP, sulfachlorpyridazine; SMR, sulfamerazine; SD, sulfadox- ine; SDZ, sulfadiazine; SQ, sulfaquinoxaline; SMZ, sulfamethazine; SMX, sulfamethoxazole; SM, sulfamoxole; SDMX, sulfadimethoxine; SIX, sulfisoxazole. ⁎ Corresponding author. Tel.: +86 22 6060 1456; fax: +86 22 6060 1332. E-mail address: s.wang@tust.edu.cn (S. Wang). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.015 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim