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Immunochemical approach for assay of herbicide thiobencarb
Analytica Chimica Acta 376 (1998) 97±101
Immunochemical approach for assay of herbicide thiobencarb Shiro Miyakea,*, Shigekazu Itob, Yuki Yamaguchia, Yoshinori Beppua, Shunichi Takewakia, Yojiro Yuasac a
Environmental Immuno-Chemical Technology Co. Ltd., Narita Research Laboratories (Iatron), Mito-aza Mitodai 1460-6, Takomachi, Katorigun, Chiba 289-22, Japan b Environmental Immuno-Chemical Technology Co. Ltd., Shizuoka Research Laboratories (Kumiai Kagaku), Kamo 3360, Kikukawa-Cho, Ogasawaragun, Shizuoka 439, Japan c Environmental Immuno-Chemical Technology Co. Ltd., Sun-Kitsukawa Bldg. 27-14, Hamamatsu-Cho, 1-Chome, Minato-Ku, Tokyo 105, Japan Received 1 June 1998; accepted 8 June 1998
Abstract Competitive enzyme-labeled immunosorbent assays (C-ELISAs) are simpler assays than instrumental analyses. Therefore, we attempted to develop a C-ELISA for assaying thiobencarb. The monoclonal antibody TBC 7-2 we produced was highly reactive with thiobencarb in this C-ELISA. Using the C-ELISA, thiobencarb-spiked water and crop samples were assayed. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Thiobencarb; Immunoassay; C-ELISA
1. Introduction Thiobencarb, S-4-chlorobenzyl N,N-diethylthiocarbamate, is a thiocarbamate herbicide (Fig. 1(A)) that has conspicuously high inter-genous selectivity between rice and barnyard grass (Echinochloa cursgalli). Because of its excellent selectivity, thiobencarb has been used widely in paddy ®elds in Japan and other countries. The monitoring of thiobencarb residues is very important to assess the safety of environment and foods. In Japan, the environmental quality standard for public and ground water is 0.02 mg/l and the water quality standard for service water is 0.02 mg/l for *Corresponding author. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00441-3
thiobencarb. The standards for foods are 0.2 ppm for rice, beans and vegetables and 0.1 ppm for wheat, barley and corn. The analysis of thiobencarb performed by gas chromatography with a ¯ame thermionic detector (GC±FTD) or gas chromatography with mass spectrometry (GC±MS) and selected ion monitoring requires several cleanup steps [1±4] and is time consuming and labor intensive. Therefore, development of a sensitive, convenient and economical method is required for analysis of the residues in a large number of environmental and food samples. Recently, immunoassays have been used as alternative or complementary methods for the analysis of pesticides because of their rapidity, sensitivity, and cost-effectiveness [5±9]. The principle of competitive immunoassay is based on a speci®c competitive
98
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
Fig. 1. Chemical structure of thiobencarb (A) and the hapten (B).
reaction between antigen (thiobencarb and thiobencarb tracer) and antibody (anti-thiobencarb antibody). This immunoassay, using an antibody speci®c to
thiobencarb, can assay this compound precisely in a crude sample and, therefore, can be used to analyze thiobencarb residues using simpler steps than instrumental analysis. Exactly, the analysis of thiobencarb residues in water by GC requires a reversed-phase (C18) solid phase extraction cleanup procedure as shown in Fig. 2(A). An immunoassay, however, can directly analyze the water sample as shown in Fig. 2(B). On the other hand, the analysis of thiobencarb residues in grains also requires 4 cleanup steps as shown in Fig. 2(C). An immunoassay of grains is also carried out easily using a sample prepared only by extraction with methanol and dilution with an adequate buffer as shown in Fig. 2(D). In this report, we describe our approach to developing a competitive enzyme-labeled immunosorbent assay (C-ELISA) for thiobencarb.
Fig. 2. Comparison of instrumental analysis and C-ELISA cleanup steps. (A) For instrumental analysis of thiobencarb in water. (B) For C-ELISA of thiobencarb in water. (C) For instrumental analysis of thiobencarb in grains. (D) For C-ELISA of thiobencarb in grains.
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
2. C-ELISA development 2.1. Monoclonal antibody production To develop an immunoassay, an antibody must ®rst be raised against an antigen of interest. However, the immune-system in vivo responds only to macromolecules, like a protein, not to small molecules, such as thiobencarb. Therefore, the host animal must be immunized with a conjugate of this small compound (hapten) and a protein (carrier protein) covalently bound. The hapten design is a very important factor in producing more speci®c antibodies.
99
Designs of the hapten for thiobencarb were shown by Shirley J. Gee et al. in 1990 [10]. However, the reactivity of the antibody with thiobencarb was a little less than our objective value which was driven by environmental and water quality standards. So, we attempted to synthesize a hapten that mimicked thiobencarb, as much as possible, with an active site to bind carrier protein as shown in Fig. 1(B). Next, we attempted to produce polyclonal and monoclonal antibodies raised against the hapten synthesized. The monoclonal antibodies selected were more reactive than the polyclonal antibodies. The monoclonal antibody TBC 7-2 was selected, based on its sensitivity
Fig. 3. Flow chart of C-ELISA for the assay of thiobencarb. (A) For samples which contain a large amount of thiobencarb. (B) For samples which don't contain thiobencarb.
100
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
with thiobencarb, from among these antibodies for further examinations. 2.2. C-ELISA construction After TBC 7-2 was obtained, we attempted to develop a C-ELISA using a conjugate of the hapten and horseradish peroxidase (HRP±hapten) as a thiobencarb tracer. As shown in Fig. 3, thiobencarb and HRP±hapten reacted competitively in the wells of microtiter plates with 96 wells coated with TBC 7-2 for 1 h at room temperature. After washing three times with buffer, the substrate solution for HRP (3,30 ,5,50 -tetramethyl-benzidine and H2O2) was added to the wells, and the plates were incubated for 10 min at room temperature. The color development (blue) was stopped by adding an equal volume of 1N H2SO4 (blue is changed to yellow by H2SO4), and the absorbance at 450 nm was measured with an automated microplate reader. When a large amount of thiobencarb was present in a sample, the absorbance was reduced (Fig. 3(A)), and when a small amount of thiobencarb was present, the absorbance was increased (Fig. 3(B)). Since the C-ELISA is constructed with a constant amount of TBC 7-2 and HRP±hapten, the absorbance is dependent on thiobencarb concentration.
Fig. 4. Standard curve of thiobencarb for C-ELISA.
2.3. Reaction of thiobencarb with TBC 7-2 A typical standard curve of thiobencarb is shown in Fig. 4. The C-ELISA could assay thiobencarb with high sensitivity; the linear concentration range was between 5 and 100 ng/ml. The success of the CELISA may be due to the fact that the immunized hapten closely mimics thiobencarb and that the TBC 7-2 monoclonal antibody was selected by carefully screening many monoclonal antibodies, based on reactivity with thiobencarb. 2.4. Spike/recovery test Even though a precise standard curve was obtained, the C-ELISA developed here had to be con®rmed by assaying thiobencarb-spiked samples of water and grains, because the matrices might have an effect on the reactivity of the antibody to the antigen. Therefore, spike/recovery tests were carried out to examine
Fig. 5. Comparison of the GC±MS and C-ELISA values for thiobencarb residues in grain samples.
application of samples prepared from water and unpolished rice. A methanol solution of thiobencarb was added to surface water collected from a pond (Tega-numa). Aliquots of this forti®ed water were assayed directly in the C-ELISA. The recovery of spiked thiobencarb (10 and 50 ppb) was very good, with the percent recoveries being between 95% and 105%. In addition, methanol solutions of thiobencarb were used to spike unpolished rice, and the samples were allowed to stand for 24 h to allow the methanol to evaporate. The forti®ed rice was ground to a meal and extracted with methanol. The methanol extract was
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
101
®ltered, and the ®ltrate was diluted with borate buffer and assayed in C-ELISA. The recovery of spiked thiobencarb (200 and 800 ppb), in each of 12 samples was also good, with the percent recoveries being between 79% and 119%. Thus, it was found that the C-ELISA for thiobencarb was satisfactory reliable.
near future, we hope that this C-ELISA will be used for the monitoring of thiobencarb residues in the environment and food.
2.5. Comparison of C-ELISA and GC±MS
[1] K. Ishikawa, R. Shinohara, K. Akasaki, Agric. Biol. Chem. 35 (1971) 1161. [2] H. Kobayashi, K. Ohyama, N. Tomiyama, Y. Jimbo, O. Matano, S. Goto, J. Chromatogr. 643 (1993) 197. [3] M.J. Redondo, M.J. Ruiz, R. Boluda, G. Font, J. Chromatogr. A 678 (1994) 375. [4] M. Takeda, Y. Ito, Y. Odanaka, K. Komatsu, Y. Maekawa, O. Matano, in: Noyaku Zanryu, Bunskiho Kenkyuhan (Eds.), Saishin Noyaku No Zanryu Bunsekiho (in Japanese), Chuo Hoki Publishing, Tokyo, 1995, 619 pp. [5] B.G. Tweedy, H.J. Dishburger, L.G. Ballantine, J. McCarthy (Eds.), Pesticide Residues and Food Safety, ACS Symposium Series 446, 1991. [6] J.O. Nelson, A.E. Karu, R.B. Wong (Eds.), Immunoanalysis of Agrochemicals, ACS Symposium Series 586, 1995. [7] R.C. Beier, L.H. Stanker (Eds.), Immunoassays for Residue Analysis, ACS Symposium Series 621, 1996. [8] J.M. Van Emon, C.L. Gerlach, J.C. Johnson (Eds.), Environmental Immunochemical Methods, ACS Symposium Series 646, 1996. [9] D.S. Aga, E.M. Thurman (Eds.), Immunochemical Technology for Environmental Applications, ACS Symposium Series 657, 1997. [10] S.J. Gee, R.O. Harrison, M.H. Goodrow, A.L. Braun, B.D. Hammock, in: M. Vanderlaan, L.H. Stanker, B.E. Watkins, D.W. Roberts (Eds.), Immunoassays for Trace Chemical Analysis, ACS Symposium Series 451, 1991, 100 pp.
The results of the spike/recovery tests carried out using unpolished rice were also con®rmed by GC±MS. The correlations of C-ELISA and GC±MS agreed well as shown in Fig. 5, although these data are preliminary, and we think that the correlations between CELISA and GC±MS from many more spike/recovery samples must be evaluated. 3. Conclusions Thiobencarb can be analyzed quantitatively in the concentration range between 5 and 100 ng/ml in this C-ELISA using the thiobencarb tracer discussed here and the TBC 7-2 antibody. The spike/recovery tests for water and unpolished rice showed that the C-ELISA for thiobencarb had good reliability. The C-ELISA, based on the hapten shown in Fig. 1 and the monoclonal antibody TBC 7-2, has potential as a simpli®ed assay method for thiobencarb, although the correlation data between GC±MS and C-ELISA are limited. In the
References
Immunochemical approach for assay of herbicide thiobencarb Shiro Miyakea,*, Shigekazu Itob, Yuki Yamaguchia, Yoshinori Beppua, Shunichi Takewakia, Yojiro Yuasac a
Environmental Immuno-Chemical Technology Co. Ltd., Narita Research Laboratories (Iatron), Mito-aza Mitodai 1460-6, Takomachi, Katorigun, Chiba 289-22, Japan b Environmental Immuno-Chemical Technology Co. Ltd., Shizuoka Research Laboratories (Kumiai Kagaku), Kamo 3360, Kikukawa-Cho, Ogasawaragun, Shizuoka 439, Japan c Environmental Immuno-Chemical Technology Co. Ltd., Sun-Kitsukawa Bldg. 27-14, Hamamatsu-Cho, 1-Chome, Minato-Ku, Tokyo 105, Japan Received 1 June 1998; accepted 8 June 1998
Abstract Competitive enzyme-labeled immunosorbent assays (C-ELISAs) are simpler assays than instrumental analyses. Therefore, we attempted to develop a C-ELISA for assaying thiobencarb. The monoclonal antibody TBC 7-2 we produced was highly reactive with thiobencarb in this C-ELISA. Using the C-ELISA, thiobencarb-spiked water and crop samples were assayed. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Thiobencarb; Immunoassay; C-ELISA
1. Introduction Thiobencarb, S-4-chlorobenzyl N,N-diethylthiocarbamate, is a thiocarbamate herbicide (Fig. 1(A)) that has conspicuously high inter-genous selectivity between rice and barnyard grass (Echinochloa cursgalli). Because of its excellent selectivity, thiobencarb has been used widely in paddy ®elds in Japan and other countries. The monitoring of thiobencarb residues is very important to assess the safety of environment and foods. In Japan, the environmental quality standard for public and ground water is 0.02 mg/l and the water quality standard for service water is 0.02 mg/l for *Corresponding author. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00441-3
thiobencarb. The standards for foods are 0.2 ppm for rice, beans and vegetables and 0.1 ppm for wheat, barley and corn. The analysis of thiobencarb performed by gas chromatography with a ¯ame thermionic detector (GC±FTD) or gas chromatography with mass spectrometry (GC±MS) and selected ion monitoring requires several cleanup steps [1±4] and is time consuming and labor intensive. Therefore, development of a sensitive, convenient and economical method is required for analysis of the residues in a large number of environmental and food samples. Recently, immunoassays have been used as alternative or complementary methods for the analysis of pesticides because of their rapidity, sensitivity, and cost-effectiveness [5±9]. The principle of competitive immunoassay is based on a speci®c competitive
98
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
Fig. 1. Chemical structure of thiobencarb (A) and the hapten (B).
reaction between antigen (thiobencarb and thiobencarb tracer) and antibody (anti-thiobencarb antibody). This immunoassay, using an antibody speci®c to
thiobencarb, can assay this compound precisely in a crude sample and, therefore, can be used to analyze thiobencarb residues using simpler steps than instrumental analysis. Exactly, the analysis of thiobencarb residues in water by GC requires a reversed-phase (C18) solid phase extraction cleanup procedure as shown in Fig. 2(A). An immunoassay, however, can directly analyze the water sample as shown in Fig. 2(B). On the other hand, the analysis of thiobencarb residues in grains also requires 4 cleanup steps as shown in Fig. 2(C). An immunoassay of grains is also carried out easily using a sample prepared only by extraction with methanol and dilution with an adequate buffer as shown in Fig. 2(D). In this report, we describe our approach to developing a competitive enzyme-labeled immunosorbent assay (C-ELISA) for thiobencarb.
Fig. 2. Comparison of instrumental analysis and C-ELISA cleanup steps. (A) For instrumental analysis of thiobencarb in water. (B) For C-ELISA of thiobencarb in water. (C) For instrumental analysis of thiobencarb in grains. (D) For C-ELISA of thiobencarb in grains.
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
2. C-ELISA development 2.1. Monoclonal antibody production To develop an immunoassay, an antibody must ®rst be raised against an antigen of interest. However, the immune-system in vivo responds only to macromolecules, like a protein, not to small molecules, such as thiobencarb. Therefore, the host animal must be immunized with a conjugate of this small compound (hapten) and a protein (carrier protein) covalently bound. The hapten design is a very important factor in producing more speci®c antibodies.
99
Designs of the hapten for thiobencarb were shown by Shirley J. Gee et al. in 1990 [10]. However, the reactivity of the antibody with thiobencarb was a little less than our objective value which was driven by environmental and water quality standards. So, we attempted to synthesize a hapten that mimicked thiobencarb, as much as possible, with an active site to bind carrier protein as shown in Fig. 1(B). Next, we attempted to produce polyclonal and monoclonal antibodies raised against the hapten synthesized. The monoclonal antibodies selected were more reactive than the polyclonal antibodies. The monoclonal antibody TBC 7-2 was selected, based on its sensitivity
Fig. 3. Flow chart of C-ELISA for the assay of thiobencarb. (A) For samples which contain a large amount of thiobencarb. (B) For samples which don't contain thiobencarb.
100
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
with thiobencarb, from among these antibodies for further examinations. 2.2. C-ELISA construction After TBC 7-2 was obtained, we attempted to develop a C-ELISA using a conjugate of the hapten and horseradish peroxidase (HRP±hapten) as a thiobencarb tracer. As shown in Fig. 3, thiobencarb and HRP±hapten reacted competitively in the wells of microtiter plates with 96 wells coated with TBC 7-2 for 1 h at room temperature. After washing three times with buffer, the substrate solution for HRP (3,30 ,5,50 -tetramethyl-benzidine and H2O2) was added to the wells, and the plates were incubated for 10 min at room temperature. The color development (blue) was stopped by adding an equal volume of 1N H2SO4 (blue is changed to yellow by H2SO4), and the absorbance at 450 nm was measured with an automated microplate reader. When a large amount of thiobencarb was present in a sample, the absorbance was reduced (Fig. 3(A)), and when a small amount of thiobencarb was present, the absorbance was increased (Fig. 3(B)). Since the C-ELISA is constructed with a constant amount of TBC 7-2 and HRP±hapten, the absorbance is dependent on thiobencarb concentration.
Fig. 4. Standard curve of thiobencarb for C-ELISA.
2.3. Reaction of thiobencarb with TBC 7-2 A typical standard curve of thiobencarb is shown in Fig. 4. The C-ELISA could assay thiobencarb with high sensitivity; the linear concentration range was between 5 and 100 ng/ml. The success of the CELISA may be due to the fact that the immunized hapten closely mimics thiobencarb and that the TBC 7-2 monoclonal antibody was selected by carefully screening many monoclonal antibodies, based on reactivity with thiobencarb. 2.4. Spike/recovery test Even though a precise standard curve was obtained, the C-ELISA developed here had to be con®rmed by assaying thiobencarb-spiked samples of water and grains, because the matrices might have an effect on the reactivity of the antibody to the antigen. Therefore, spike/recovery tests were carried out to examine
Fig. 5. Comparison of the GC±MS and C-ELISA values for thiobencarb residues in grain samples.
application of samples prepared from water and unpolished rice. A methanol solution of thiobencarb was added to surface water collected from a pond (Tega-numa). Aliquots of this forti®ed water were assayed directly in the C-ELISA. The recovery of spiked thiobencarb (10 and 50 ppb) was very good, with the percent recoveries being between 95% and 105%. In addition, methanol solutions of thiobencarb were used to spike unpolished rice, and the samples were allowed to stand for 24 h to allow the methanol to evaporate. The forti®ed rice was ground to a meal and extracted with methanol. The methanol extract was
S. Miyake et al. / Analytica Chimica Acta 376 (1998) 97±101
101
®ltered, and the ®ltrate was diluted with borate buffer and assayed in C-ELISA. The recovery of spiked thiobencarb (200 and 800 ppb), in each of 12 samples was also good, with the percent recoveries being between 79% and 119%. Thus, it was found that the C-ELISA for thiobencarb was satisfactory reliable.
near future, we hope that this C-ELISA will be used for the monitoring of thiobencarb residues in the environment and food.
2.5. Comparison of C-ELISA and GC±MS
[1] K. Ishikawa, R. Shinohara, K. Akasaki, Agric. Biol. Chem. 35 (1971) 1161. [2] H. Kobayashi, K. Ohyama, N. Tomiyama, Y. Jimbo, O. Matano, S. Goto, J. Chromatogr. 643 (1993) 197. [3] M.J. Redondo, M.J. Ruiz, R. Boluda, G. Font, J. Chromatogr. A 678 (1994) 375. [4] M. Takeda, Y. Ito, Y. Odanaka, K. Komatsu, Y. Maekawa, O. Matano, in: Noyaku Zanryu, Bunskiho Kenkyuhan (Eds.), Saishin Noyaku No Zanryu Bunsekiho (in Japanese), Chuo Hoki Publishing, Tokyo, 1995, 619 pp. [5] B.G. Tweedy, H.J. Dishburger, L.G. Ballantine, J. McCarthy (Eds.), Pesticide Residues and Food Safety, ACS Symposium Series 446, 1991. [6] J.O. Nelson, A.E. Karu, R.B. Wong (Eds.), Immunoanalysis of Agrochemicals, ACS Symposium Series 586, 1995. [7] R.C. Beier, L.H. Stanker (Eds.), Immunoassays for Residue Analysis, ACS Symposium Series 621, 1996. [8] J.M. Van Emon, C.L. Gerlach, J.C. Johnson (Eds.), Environmental Immunochemical Methods, ACS Symposium Series 646, 1996. [9] D.S. Aga, E.M. Thurman (Eds.), Immunochemical Technology for Environmental Applications, ACS Symposium Series 657, 1997. [10] S.J. Gee, R.O. Harrison, M.H. Goodrow, A.L. Braun, B.D. Hammock, in: M. Vanderlaan, L.H. Stanker, B.E. Watkins, D.W. Roberts (Eds.), Immunoassays for Trace Chemical Analysis, ACS Symposium Series 451, 1991, 100 pp.
The results of the spike/recovery tests carried out using unpolished rice were also con®rmed by GC±MS. The correlations of C-ELISA and GC±MS agreed well as shown in Fig. 5, although these data are preliminary, and we think that the correlations between CELISA and GC±MS from many more spike/recovery samples must be evaluated. 3. Conclusions Thiobencarb can be analyzed quantitatively in the concentration range between 5 and 100 ng/ml in this C-ELISA using the thiobencarb tracer discussed here and the TBC 7-2 antibody. The spike/recovery tests for water and unpolished rice showed that the C-ELISA for thiobencarb had good reliability. The C-ELISA, based on the hapten shown in Fig. 1 and the monoclonal antibody TBC 7-2, has potential as a simpli®ed assay method for thiobencarb, although the correlation data between GC±MS and C-ELISA are limited. In the
References