Molecular and Immunochemical Characteristics of Monoclonal and Recombinant Antibodies Selective for the Triazine Herbicide Simetryn and Application to Environmental Analysis KOSUKE NISHI,* ,†,‡ MASATAKE ISHIUCHI, † KOSUKE MORIMUNE, § AND HIDEO OHKAWA † Research Center for Environmental Genomics, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan, and Naruto Research Center, Otsuka Chemical Company, Limited, 615 Hanamen, Satoura-cho, Naruto, Tokushima 772-0021, Japan A monoclonal antibody (mab) selective for the thiomethyl-s-triazine herbicide simetryn was obtained and characterized in enzyme-linked immunosorbent assay (ELISA). An IC 50 value for simetryn was 8.5 ng/mL, and the detection range extended from 1.1 to 70 ng/mL in ELISA. The cDNAs encoding variable heavy chain (VH) and variable light chain (VL) of the mab were cloned to produce various recombinant antibodies. Single-chain variable fragment (scFv) antibodies derived from the mab were characterized in ELISA and showed similar reactivities and specificities to the parent mab. A urea denaturation test revealed that the scFv antibodies bound to simetryn were more stable than those in the absence of antigen. A sandwich ELISA based on VH and VL fragments of the mab was successfully developed and showed similar sensitivity to those based on the mab and scFv antibodies in ELISA. In the recovery experiments using spiked environmental samples, the results obtained in ELISA based on the mab were favorably correlated with those by HPLC. KEYWORDS: ELISA; simetryn; triazine herbicide; monoclonal antibody; scFv INTRODUCTION The triazine group of herbicides is the most well-known and has been widely used for more than 40 years in the world. These chemicals inhibit photosynthesis in plants by blocking the electron transfer at the reducing site of chloroplast photosystem II electron transport (1). The thiomethyl-s-triazine herbicide simetryn introduced in 1969 in Japan is one of the principal herbicides used for control of broad-leaved weeds in paddy fields. Paddy fields for rice cultivation make up 55% of total farmland in Japan (2), which are some of the most important sources of environmental water pollution by pesticides because of easy runoff into rivers after the application (3). Simetryn has relatively high water solubility (450 mg/L) (4) and is of concern with respect to the influence to human health and aquatic organisms (5, 6). Therefore, it is necessary to know the actual concentration levels of simetryn in the surface water. Quantification of simetryn residues in environmental samples has been carried out by instrumental methods such as gas chromatography (5, 6) or high-performance liquid chromatog- raphy (HPLC) (7). However, these methods require time- consuming cleanup steps and expensive equipments. On the other hand, immunoassay is a cost-effective and relatively simple method that can handle a lot of samples in a short time. The use of monoclonal antibody (mab) has some advantages such as the homogeneity of affinity and specificity to an antigen. Moreover, cloning and expression methods of antibody genes have opened the opportunities for antibody engineering (8). Production of recombinant antibodies in bacterial culture is even readily available and less expensive than that of mabs in animal cell culture. Gene manipulation is also available to alter the property of recombinant antibodies. Triazine herbicides such as atrazine are one of the most extensively investigated groups for environmental analysis by immunoassays and for develop- ment of recombinant antibodies (9-13). In this paper, we attempted (1) to produce anti-simetryn monoclonal and recombinant antibodies, (2) to investigate the molecular and immunochemical characteristics of the antibodies in enzyme-linked immunosorbent assay (ELISA), and (3) to validate the ELISA system for the detection of simetryn residues in the environmental water samples. MATERIALS AND METHODS Chemicals and Biochemicals. Simetryn [2,4-bis(ethylamino)-6- methylthio-1,3,5-triazine] (Figure 1) and other triazine herbicides were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Simetryn haptens S1 and S2 (Figure 1) were synthesized in Otsuka Chemical Co., Ltd. (Tokushima, Japan). Rabbit serum albumin (RSA), bovine serum * To whom correspondence should be addressed. Telephone: (530) 754- 8884. Fax: (530) 752-1537. E-mail: knishi@ucdavis.edu. † Kobe University. ‡ Present address: Department of Entomology, University of California, One Shields Avenue, Davis, CA 95616. § Otsuka Chemical Co., Ltd. 5096 J. Agric. Food Chem. 2005, 53, 5096-5104 10.1021/jf050246t CCC: $30.25 © 2005 American Chemical Society Published on Web 06/01/2005