Analytica Chimica Acta 491 (2003) 1–13 Production of antibodies and development of enzyme-linked immunosorbent assays for the herbicide butachlor Julia Yakovleva a , Anatoly V. Zherdev a , Valentina A. Popova b , Sergei A. Eremin b , Boris B. Dzantiev a,∗ a Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, 119071 Moscow, Russia b Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119899 Moscow, Russia Received 6 March 2003; received in revised form 10 June 2003; accepted 25 June 2003 Abstract Specific polyclonal antibodies against the herbicide butachlor [2-chloro-N-(2,6-diethylphenyl)-N-(butoxymethyl)acetani- lide] were obtained from rabbits immunized with butachlor-3-mercaptopropionic acid (BMPA), coupled to bovine serum albumin (BSA) and soybean trypsin inhibitor. Competitive enzyme-linked immunosorbent assays with photometric detection based on either immobilized antibody (direct assay) or immobilized antigen (indirect assay) were developed and optimized for quantification of butachlor. The best assay sensitivity was observed using antibodies against BMPA–BSA immunogen and immobilized antigen format, where the limit of detection was 0.02 ng ml -1 , the standard curve midpoint (IC 50 ) was 2.4 ng ml -1 and assay duration was 1.5 h. Antibodies were capable of the specific detection of butachlor with cross-reactivities towards structurally related chloroacetanilide herbicides (alachlor, acetochlor, dimethachlor, metolachlor, propachlor) below 0.3%. Direct and indirect assays are compatible with up to 1–2%, v/v of methanol and ethanol in the analyzed probe. Assay application to the analysis of mineral, ground and surface water resulted in butachlor recoveries varying between 70 and 118% between 10 and 100 ng ml -1 herbicide concentrations. © 2003 Elsevier B.V. All rights reserved. Keywords: Butachlor; Chloroacetanilides; Enzyme immunoassay; Cross-reactivity; Environmental water analysis 1. Introduction Intensive use of agrochemicals has resulted in the pollution of the biosphere with toxic compounds. Nowadays pesticides are one of the most prevalent environmental pollutants. Detection of pesticides and their metabolites in environmental water is an important task, especially for ground water that is used for drinking purposes. Among with traditional physico-chemical methods based on gas or liquid ∗ Corresponding author. Tel./fax: +7-095-954-2804. E-mail address: bdzan@online.ru (B.B. Dzantiev). chromatography, immunoassay technology is increas- ingly used for screening of pesticides due to its sen- sitivity, selectivity, time efficiency and portability [1]. Butachlor [2-chloro-N-(2,6-diethylphenyl)-N-(but- oxymethyl)acetanilide] is a recently registered pre-em- ergence herbicide belonging to the chloroacetanilide family and it is widely used to control grasses in rice crops. Despite the high rates of butachlor application, there is little information available about the occur- rence of this herbicide in the environment. Butachlor has a moderate persistence in soil, but persists for a long time in water. For instance, in China, where butachlor is one of the predominating herbicides in 0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00796-7