Analytica Chimica Acta 682 (2010) 93–103 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Hapten synthesis, monoclonal antibody generation, and development of competitive immunoassays for the analysis of picoxystrobin in beer Francesc A. Esteve-Turrillas a,1 , Javier Parra a,1 , Antonio Abad-Fuentes a , Consuelo Agulló b , Antonio Abad-Somovilla b , Josep V. Mercader a,∗ a Department of Biotechnology, IATA–CSIC, Agustí Escardino 7, 46980 Paterna, València, Spain b Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100 Burjassot, València, Spain article info Article history: Received 30 July 2010 Received in revised form 22 September 2010 Accepted 26 September 2010 Available online 8 October 2010 Keywords: ELISA Pesticide Hapten synthesis Protein coupling Cereal Food analysis abstract This paper describes the original synthesis of a functionalized derivative of the fungicide picoxystrobin and the generation of the first reported monoclonal antibodies against this strobilurin pesticide. The syn- thetic hapten was prepared by total synthesis from commercial chemicals and incorporating the spacer arm through a carbon–carbon single bond. Also, to obtain the immunogen, an uncommon hapten activa- tion strategy based on N,N ′ -disuccinimidyl carbonate was employed, affording high activation yields and clean and reproducible coupling results. With these immunoreagents, two enzyme-linked immunosor- bent assays (ELISAs) were developed: a competitive one-step assay using the antibody-coated direct ELISA format and a competitive two-step assay with the conjugate-coated indirect ELISA procedure. Both immunoassays were characterized in terms of sensitivity, selectivity, tolerance to solvents and matrix effects, achieving limits of detection below 0.2 gL -1 . The optimized assays were used for the determi- nation of picoxystrobin residues in beer, with recovery values ranging between 90 and 121% for the direct assay and from 79 to 122% for the indirect assay. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Strobilurin pesticides are synthetic analogues of the - methoxyacrylate compounds produced by the fungus Strobilurus tenacellus and Oudemansiella mucida. Among all of them, picoxys- trobin (PC) and azoxystrobin are the two only synthetic strobilurin fungicides that retain the natural toxophore group [1]. Particularly, PC was first announced by Syngenta in 2000, although it is cur- rently commercialized by DuPont Crop Protection under the brand name of Acanto as a suspension concentrate containing 23% (w/w) of active principle. PC is employed for cereal crop protection, par- ticularly wheat, barley and oat crops, against yellow, brown and crown rust caused by basidiomycetes of the genus Puccinia, and Abbreviations: BSA, bovine serum albumin; CB, carbonate buffer; CR, cross- reactivity; DMF, N,N-dimethylformamide; DSC, N,N ′ -disuccinimidyl carbonate; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidise; IA, immunoassay; LOD, limit of detection; LOQ, limit of quantitation; mAb, mono- clonal antibody; MR, molar ratio; MRL, maximum residue limit; OVA, ovalbumin; PB, phosphate buffer; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween 20; PBT, phosphate buffer with Tween 20; PC, picoxystrobin; TBST, Tris-buffered saline with Tween 20. ∗ Corresponding author. Tel.: +34 963900022; fax: +34 963636301. E-mail address: jvmercader@iata.csic.es (J.V. Mercader). 1 1 Both of Francesc A. Esteve-Turrillas and Javier Parra rank the first authors. against septoria blotch produced by Septoria tritici (teleomorph Mycosphaerella graminicola), net blotch induced by Pyrenophora teres and powdery mildew provoked by ascomycetes of the genus Blumeria [2,3]. PC is one of the fungicides accepted by the British Beer and Pub Association (BBPA) and the Brewing Research Inter- national (BRI) for use on barley and wheat intended for malting and brewing [4]. The extensive and intensive uses of agrochemical compounds to fight and prevent diseases in all kinds of crops are the main rea- son for residual amounts of these toxic substances being commonly found in food products. Consequently, the regulatory authorities of most countries have defined maximum residues levels (MRLs) for all pesticides in food and feed commodities. The European Regula- tion no. 396/2005 has established the MRL for PC at 0.05 mg kg -1 for all the food groups, with the exception of barley and oats for which a MRL of 0.20 mg kg -1 was set up [5]. Many studies have demon- strated that residues of pesticides can remain in the malt after the mashing and boiling processes, mainly those with octanol–water coefficients (K OW ) between 2 and 4 (the K OW for PC is 3.6) [6]. In fact, pendimethalin, trifluralin, fenitrothion, malathion, myclobu- tanil, nuarimol, propiconazole, carbaryl and demethomorph have been found in malt and malt beverages [7,8]. To determine PC in food samples, gas [9,10] or liquid [11,12] chromatographic separation techniques coupled to a mass spectrometry detector are usually involved. However, chromatographic methodologies 0003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2010.09.042