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Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb
Available online at www.sciencedirect.com Agricultural Sciences i n China 2007, 6 ( 2 ) : 175-179
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February 2007
Production and Characterization of Polyclonal Antibody for the NMethylcarbarnate Insecticide Metolcarb ZHANG Qil, LI Tie-junl, ZHU Xiao-xial, XU Li-nal, LIU Feng-quanl,HU Bai-shi', JIANG Ying-hua1 and
CAO Bin2 I
Key Laboratory of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture/Department of Plant Protection, Nunjing Agricultural University, Nunjing 2 10095, P.R. China
2
Pesticide Research Institute of Jiangsu Province, Nanjing 210036, P. R. China
Abstract The hapten, 3-{[ 1 -(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 'H-nuclear magnetic resonance spectrometry ('H-NMR). Then, HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimidddicyclohexylcarbodimide (NHSDCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAbOl reached 1.28 x 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAbOl with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.
Key words: metolcarb, antigen, polyclonal antibody, antiserum characterization, ELISA
INTRODUCTION Metolcarb, 3-methylphenyl methylcarbamate, is one of the N-methylcarbamate insecticides. The analytical methods currently used for the detection of metolcarb, such as gas chromatography (GC) (Lin et al. 2003) and high-pressure liquid chromatography (HPLC), mainly involve conventional instruments (Fang et al. 1997). These methods, however, involving multistep procedures for sample cleanup, are expensive and time consuming, and require manual intervention and large amounts of organic solvents. The current methods can be used only in laboratories, with only trained personnel being capable of using these instruments. Recently, immunological methods that
have been developed to detect small molecular compounds make it possible to establish a new protocol for the detection of metolcarb. An immunoassay would be a fast, simple, and sensitive method and can especially meet the need of detection of multiple samples at one point of time and onsite monitoring (Lopez et al. 1996; Hennion and Barcelo 1998). There are several rapid immunoassay methods that have been reported for N-methylcarbamate insecticides, such as aldicarb (Brady et al. 1989), carbofuran (Jourdan et al. 1995; Liu et a1. 2000), carbaryl (Marco et al. 1993; Abad and Montoya 1994; Liu et al. 1999), methiocarb (Abad et al. 1998), and propoxur (Moreno et al. 2001). But thus far, it is not reported for production of metolcarb antibody. In this paper, good polyclonal antibody for metolcarb was first prepared, which paved
Received 26 June, 2006 Accepted 13 October, 2006 ZHANG Qi, Ph D candidate, E-mail: changqi52lx @ 126.com; Correspondence LIU Fmg-quan, Professor, Tel: +86-25.84396726, E-mail: fqliu20011 @sina,com.cn
02007,CAAS. All rights reserved. Published by Elsevier Ltd.
ZHANG Qi et al.
176
the way for development of enzyme-linked immunosorbent assay (ELISA) for metolcarb.
MATERIALS AND METHODS Chemicals, instruments, and buffer solutions The starting materials, 97% of m-cresol, trichloromethyl carbonochloridate, and p-alanine, for hapten synthesis were purchased from Acros Organics in USA and Shanghai Chemicals Co. in China. Standards of metolcarb and other N-methylcarbamate insecticides were supplied by Pesticide Research Center of Jiangsu Province, China. N-Hydroxysuccinimide(NHS), N,Ndicyclohexylcarbodimide (DCC), bovine serum albumin (BSA), ovalbumin (OVA), and Freunds complete and incomplete adjuvant were purchased from Sigma Chemical Co., USA. 3,3 ',5,5'-tetramethylbenzidine (TMB) and goat antirabbit (GAR) immunoglobulin conjugated to horseradish peroxidase (HRP) were purchased from Huamei Biotechnology Co., China. ELISA experiments were performed in 96-well microplates (Corning, USA), and the absorbances were measured with a DNM-9602 microplate reader (Prolong New Technology Co.,Beijing, China). UV-VIS spectra for the verification of conjugates were obtained using a spectrophotometer (DU-640 Beckman, USA). Infrared spectra were obtained using FI-IR (Nicolet, USA). Mass spectra were obtained on QP 5050 GC-MS spectrometer (Shimadzu, JAPAN). NMR spectra were obtained using a general electric ACF-300 MHz spectrometer (Brucker, Germany). Normal strength phosphate-buffered saline (1 x PBS: 8 g L-I NaCI, 1.15 g L-I Na,HPO,, 0.2 g L-' M,PO,, 0.2 g L-' KC1, pH 7 3 , PBST (1xPBS containing 0.05% (v/v) Tween 20, pH 7 . 3 , carbonate buffer (1.59 g L-I Na,CO,, 2.93 g L-I NaHCO,, pH 9.6), citric acid-phosphate buffer (pH 5.0) were used for the immunoassay.
Hapten synthesis and verification The hapten of metolcarb, 3-{[l-(3-(methy1)phenyloxy) carbonyl]amino}propanoic acid (HOM), was synthesized as shown schematically in Fig.1. (Phosgene is a highly toxic gas that necessitates careful handling and
Fig. 1 Synthetic scheme for the hapten HOM.
the use of a well-ventilated fume hood). Finally, 1.9 g of the pure hapten HOM was obtained, with recovery of product being 48% (it was calculated from m-cresol). Spectra of the hapten HOM were analyzed as follows: IR: 3 287 (s, N-H), 3 047 (m, Ar-H), 2 813-2942 (s-m, C-H), 2770-2500 (w, 0-H), 1704 (s, G O ) , 1544 (s, d&, 1488, 1440 (m, dCH,), 1237 (s, Ar-0-C), 1075 (m, C-0, carboxyl acid). In addition, only one small peak was obtained between 700 and 800 cm-I, which indicates that two sites (I-th and 3-th) on the aromqtic ring of HOM were replaced. EI: 43 (-CONH- or -NHCH,CH,-), 45 (-COOH), 56 (-CH,CH,OC-), 60 (-OOCNH-), 77 (C,H,+H+), 91 [-C,H,(CH,)], 107 (CH,C,H,O-), 121 (-C,H,OOC+ H+), 206 [C,H,(CH,) COONHC%CH,CO-], 223 (HOM'). 'H-NMR (DMSO): 6 2.35 (s, 3H, ArCH, ), 2.46-2.55 (t, 2H, HOOC-CH,), 3.50-3.61 (p, 2H, N-CH,), 5.66 (s, IH, N-H), 6.647.28 (p, 4H, Ar-H).
Preparationof hapten-proteinconjugates The hapten HOM was conjugated with proteins (BSA and OVA) using the activated ester method (AE method). One micromole of the hapten was incubated overnight with stimng at room temperature with stoichiometric amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide in 1500 pL of DMF. The mixture was cooled to 5°C for 2 h. After centrifugation, 550 pL of the clear supernatant containing the active BSA ester was added slowly to 2.5 mL, of a 12 mg d-' solution in 0.2 mol L-' phosphate buffer, pH 8.0 (another
Q2007,CMS. All rights resewed. Publishedby Elsevier Lid.
Production and Characterization of Polyclonal Antibodv for the N-Methylcarbamatc Insecticide Mclolcarb
550 yL of the clear supernatant was added to 2.5 mL of a 12 mg mL-’ OVA solution). These two mixtures were allowed to react at room temperature for 4 h with stirring, and finally the conjugates were pullfied by dialyzing for 60 h using 0.2 mol L-’ phosphate buffer, pH 6.8. Conjugate formation was confirmed spectrophotometrically. UV-visible spectra showed qualitative differences between the carrier proteins and conjugates in the region of maximum absorbance of the hapten (256 nm). The hapten to protein molar ratio of conjugates was then estimated from the spectral data of the hapten, the protein, and the corresponding conjugate.
Immunization Two female New Zealand white rabbits were used for raising polyclonal antibodies. One milligram (protein equivalent) of the immunogen (HOM-BSA) was thoroughly emulsified with an equal volume of Freund’s adjuvant. Freund’s complete adjuvant was used for the first injection, and Freund’s incomplete adjuvant was used for the subsequent booster injections. Booster injections were given at 3-week intervals. On the 7th day after each booster injection, 5 mL blood samples were taken from an ear vein to check the titer of the antiserum. The blood samples were allowed to coagulate for 2 h at 37°C and then kept in a refrigerator overnight. The serum was centrifuged at 4°C for 10 min, and the supernatant was dispensed into 1.5-mL eppendorf tubes and stored at -20°C. Booster injections were administered six times. Finally, two antisera for metolcarb, named pAbOl and pAb02, were obtained from the rabbits’ heart blood.
1 I1
per well) was added and incubated for 1 h at 37°C. The plates were washed again, and 50 yL per well of TMB solution (3.3 pL of 30% H,O,, 400 yL of 0.6% TMR in DMSO per 25 mL of acetate buffer) was added. The color development was stopped after 10-15 min by the addition of 2 mol L H,SO, (25 pL per well). The absorbance was measured at 450 nm.
Antiserum characterization Affinity and specificity are two most important characteristics that determine antiserum quality and are expressed by the titer of an antiserum and the cross-reactivity separately. Affinity: The affinity of the antiserum collected from two rabbits was detected by incompetitive ELISA, and expressed the titer was expressed as the multiple of dilution. The higher the titer, the stronger is the affinity. Therefore, the antiserum with higher titer was chosen for further research. Specificity: The cross-reactivity not only determines the specificity of the antibody but also the reliability of the assay. Data were obtained from standard curves of metolcarb, N-methylcarbamates dimethacarb, propoxur, fenobucarb, isoprocarb, carbofuran, pirimicarb, and methomyl. Each compound was prepared in 5 % methanol in PBST and tested in the concentration range 1-1000000 ng mL-’. The cross-reactivities (CR) were calculated as a ratio of the IC,,, of metolcarb to that of the tested compound. CR was set at 100% for metolcarb.
RESULTS AND DISCUSSION
Enzyme-linked immunosorbent assay (ELISA) Microplates were coated overnight at 4°C with 50 pL per well of the appropriate coating antigen concentration in carbonate-bicarbonate buffer. After the coated plates were washed with PBST, the surface of the wells was blocked with 100 yL of 1% glutin in PBS (or 1% OVA, 3% skimmed milk powder) for 1 h at 37°C. After one step of washing, 25 pL of antiserum diluted in PBS and 25 pL of analyte solution were added per well and incubated for 1 h at 37°C. After another step of washing, goat antirabbit HRP (1:2000 in PBST, 50 pL
Synthesis of hapten and its conjugation to carrier proteins A hapten mimicking the target analyte and containing reactive groups for conjugation to carrier proteins must be designed. In this article, the hapten synthesis was aimed at preserving the metolcarb structure for electronic and hydrophobic properties and considering the ideal spacer position to elicit antibodies for metolcarb. On the basis of this idea, the hapten of metolcarb (HOM) was synthesized. The synthesis of HOM was carried
02007. CAAS Ail rights reserved Published by EisevierLtd.
ZHANG Qi et al.
17X
out as shown schematically in Fig. 1. The hapten HOM with a carboxyl terminal in its structure was conjugated to carrier proteins using the active ester method. In the ultraviolet-visible spectra obtained from continuous wavelength scanning, there were several obvious changes between the spectra of the conjugate and its carrier protein. By assuming that the molar absorptivity of haptens was the same for the free and conjugated forms, the apparent molar ratios were estimated as 23 and 17 for proteins BSA and OVA, respectively (the spectral data were obtained at 256 nm).
Antiserum characterization Affinity: The antiserum titers of terminal bleeds from two rabbits (antiserum pAbOl and pAb02) were measured under identical conditions and using identical analytical plates. The results showed that both immunized individuals could produce high titer antibody, that is to say, the antibodies we produced had quite strong affinity. The titer of pAbOl is 1.28x lo6higher than that of pAbO2 (Fig.2). To identify systems that yielded the highest sensitivity for metolcarb, competitive inhibition experiments were conducted and lowest IC,, values were chosen as the optimal combinations. Results showed that the IC,, values of the AbOl and pAbO2 systems were 281.22 and 468.54 ng mL-', respectively. Therefore, the antiserum pAbOl that was not purified by IgG protein was used in this study to test the specificity of antiserum. Specificity (cross-reactivities, CR): The antiserum pAbOl showed only few CR with dimethacarb (28.2%). Because the molecular structure of dimethacarb and -c
-&..-
pAb02 pAb0l
10
2
08 06
3 '0
- 1
-
-
5
' - 6
2 3 4
.
7
___ 8 ..
20 0
0
I
10
102
103
lo*
105
106
Concentration ofmetolcarh (ng mL ')
Fig. 3 Specificity of the optimized ELISA for metolcarb. Cross reactivity values, calculated as (Isn of metolcaMI,,, of other compound) x 100, were as follows: ( I ) metolcarb, 100.0%; (2) dimethacarb, 28.2%; (3) propoxur, 0.0%; (4) fenobucarb, 0.0%; ( 5 ) isoprocarb, 0.0%; (6) carbofuran, 0.0%; (7) pirimicarb, 0.0%; (8) methomyl, 0.0%.
metolcarb is very similar, the molecule of dimethacarb had one more methyl group on the aromatic ring compared with that of metolcarb. However, for the other six N-methylcarbamate pesticides such as propoxur, the cross-reactivity rate of this method is below 0.1% (Fig.3), that is to say, there is no cross-reactivity between these pesticides and pAbO1, indicating that the antiserum pAbOl has a high specificity to metolcarb.
CONCLUSIONS Metolcarb has been widely used for controlling pests in many crops. ELISA methods that were used for rapid detection and onsite monitoring of pesticide residue are remarkable because of their several advantages. In this study, polyclonal antibody for metolcarb with strong affinity and high specificity was produced. The results showed that if ELISA conditions (such as pH value and ionic strength) are optimized, the antibody pAbOl can be used to develop a sensitive and a rapid detection protocol for metolcarb residue.
9 Q 0
04
Acknowledgements
02
0
I
4
16
64
256
512
Dilution ol' serum ( x I0 000 times)
Fig. 2 Titres of antisera against HOM-BSA.
1024
This study was supported by the National High Technology Research and Development Program (863 Program), China (2006aalOz447) and Important Technology in Agricultural Frame Adjusting of Ministry of Agriculture, China (2003-07-02A).
02007,CAAS. All rights reserved. Publishedby Elsevier Ltd.
Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb
References Abad A, Montoya A. 1994. Production of monoclonal antibodies for carbaryl from a hapten preserving the carbamate group. Journal of Agricultural Food and Chemistry, 42, I8 18-1823. Abad A, Moreno M J, Montoya A. 1998. Hapten synthesis and production of monoclonal antibodies to the N methylcarbamate pesticide methiocarb. Journal of Agricultural Food and Chemistry, 46,2417-2426. Brady J F, Heeker J R, Wilson R A, Mumma R 0.1989. Enzymeimmunoassay for aldicarb. In: Wang R G M, Franklin C A, Honeycut R C, Reinhert J C, eds, Biological Monitoring of Pesticide Exposure, Measurement, Estimation and Risk Reduction. ACS Symposium Series, American Chemical Society, Washtionton DC. pp. 382, 262-284. Fang J F, Wang G S, Fang L, Sun W P, Jiang H P. 1997. Studies on GC assay for a formulation containing three active ingredients (metolcarb, isoprocarb and buprofezin). Chinese Journal of Zhejiang Chemical Engineering, 28,42-43. (in Chinese) Hennion M C, Barcelo D. 1998. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: a review. Analytica ChimicaActa, 362,3-34. Jourdan S W,Scutellaro A M, Fleeker J R, Herzog D P, Rubio F M. 1995. Determination of carbofuran in water and soil by a rapid magnetic particle-based ELISA. Journal of Agricultural
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Food and Chemistry, 43,2784-2788. Lin W X, Tian M, Li J Y, Cao D M. 2003. High-performance liquid chromatographic determination of N-methylcarbamate pesticides in rice by two cartridges ofsolid-phase extraction clean up. Journal of the Chinese Cereals and OilsAssociation, 18,79-84. (in Chinese) Liu S Z, Feng D H, Chen M J, Qian C F. 2000. Study on a highly specific enzyme linked immunosorbent assay for cabofuran. Chinese Journal of Analytical Science, 16, 373-378. (in Chinese) Liu S Z, Feng D H, Qian C F. 1999. Studies on enzyme linked immunosorbent assay for the determination of carbaryl. Chinese Journal of Pesticide Science, 1,62-68. (in Chinese) Lopez A V, Charan C, van Emon J. 1996. Supercritical fluid extraction - enzyme-linked immunosorbent assay applications for determination of pesticides in soil and food. In: Beier R C, Stanker L H, eds, Immunoassays for Residue Analysis: Food Safety. ACS Symposium Series, American Chemical Society, Washington DC. pp. 621,439-449. Marc0 M P, Gee S J, Cheng H M, Liang 2 Y,Hammock B D. 1993. Development of an ELISA for carbaryl. Journal of Agricultural Food and Chemistry, 41,423-430. Moreno M J, Abad A, Montoya A. 2001. Production of monoclonal antibodies to the N-methylcarbamate pesticide pmpoxur. Journal of Agricultural Food and Chemistry, 49, 72-78. (Edited by WANG Lu-han)
Oxx)7, CAAS. All rightsresewed. Publishedby ElsevierLtd.
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February 2007
Production and Characterization of Polyclonal Antibody for the NMethylcarbarnate Insecticide Metolcarb ZHANG Qil, LI Tie-junl, ZHU Xiao-xial, XU Li-nal, LIU Feng-quanl,HU Bai-shi', JIANG Ying-hua1 and
CAO Bin2 I
Key Laboratory of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture/Department of Plant Protection, Nunjing Agricultural University, Nunjing 2 10095, P.R. China
2
Pesticide Research Institute of Jiangsu Province, Nanjing 210036, P. R. China
Abstract The hapten, 3-{[ 1 -(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 'H-nuclear magnetic resonance spectrometry ('H-NMR). Then, HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimidddicyclohexylcarbodimide (NHSDCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAbOl reached 1.28 x 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAbOl with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.
Key words: metolcarb, antigen, polyclonal antibody, antiserum characterization, ELISA
INTRODUCTION Metolcarb, 3-methylphenyl methylcarbamate, is one of the N-methylcarbamate insecticides. The analytical methods currently used for the detection of metolcarb, such as gas chromatography (GC) (Lin et al. 2003) and high-pressure liquid chromatography (HPLC), mainly involve conventional instruments (Fang et al. 1997). These methods, however, involving multistep procedures for sample cleanup, are expensive and time consuming, and require manual intervention and large amounts of organic solvents. The current methods can be used only in laboratories, with only trained personnel being capable of using these instruments. Recently, immunological methods that
have been developed to detect small molecular compounds make it possible to establish a new protocol for the detection of metolcarb. An immunoassay would be a fast, simple, and sensitive method and can especially meet the need of detection of multiple samples at one point of time and onsite monitoring (Lopez et al. 1996; Hennion and Barcelo 1998). There are several rapid immunoassay methods that have been reported for N-methylcarbamate insecticides, such as aldicarb (Brady et al. 1989), carbofuran (Jourdan et al. 1995; Liu et a1. 2000), carbaryl (Marco et al. 1993; Abad and Montoya 1994; Liu et al. 1999), methiocarb (Abad et al. 1998), and propoxur (Moreno et al. 2001). But thus far, it is not reported for production of metolcarb antibody. In this paper, good polyclonal antibody for metolcarb was first prepared, which paved
Received 26 June, 2006 Accepted 13 October, 2006 ZHANG Qi, Ph D candidate, E-mail: changqi52lx @ 126.com; Correspondence LIU Fmg-quan, Professor, Tel: +86-25.84396726, E-mail: fqliu20011 @sina,com.cn
02007,CAAS. All rights reserved. Published by Elsevier Ltd.
ZHANG Qi et al.
176
the way for development of enzyme-linked immunosorbent assay (ELISA) for metolcarb.
MATERIALS AND METHODS Chemicals, instruments, and buffer solutions The starting materials, 97% of m-cresol, trichloromethyl carbonochloridate, and p-alanine, for hapten synthesis were purchased from Acros Organics in USA and Shanghai Chemicals Co. in China. Standards of metolcarb and other N-methylcarbamate insecticides were supplied by Pesticide Research Center of Jiangsu Province, China. N-Hydroxysuccinimide(NHS), N,Ndicyclohexylcarbodimide (DCC), bovine serum albumin (BSA), ovalbumin (OVA), and Freunds complete and incomplete adjuvant were purchased from Sigma Chemical Co., USA. 3,3 ',5,5'-tetramethylbenzidine (TMB) and goat antirabbit (GAR) immunoglobulin conjugated to horseradish peroxidase (HRP) were purchased from Huamei Biotechnology Co., China. ELISA experiments were performed in 96-well microplates (Corning, USA), and the absorbances were measured with a DNM-9602 microplate reader (Prolong New Technology Co.,Beijing, China). UV-VIS spectra for the verification of conjugates were obtained using a spectrophotometer (DU-640 Beckman, USA). Infrared spectra were obtained using FI-IR (Nicolet, USA). Mass spectra were obtained on QP 5050 GC-MS spectrometer (Shimadzu, JAPAN). NMR spectra were obtained using a general electric ACF-300 MHz spectrometer (Brucker, Germany). Normal strength phosphate-buffered saline (1 x PBS: 8 g L-I NaCI, 1.15 g L-I Na,HPO,, 0.2 g L-' M,PO,, 0.2 g L-' KC1, pH 7 3 , PBST (1xPBS containing 0.05% (v/v) Tween 20, pH 7 . 3 , carbonate buffer (1.59 g L-I Na,CO,, 2.93 g L-I NaHCO,, pH 9.6), citric acid-phosphate buffer (pH 5.0) were used for the immunoassay.
Hapten synthesis and verification The hapten of metolcarb, 3-{[l-(3-(methy1)phenyloxy) carbonyl]amino}propanoic acid (HOM), was synthesized as shown schematically in Fig.1. (Phosgene is a highly toxic gas that necessitates careful handling and
Fig. 1 Synthetic scheme for the hapten HOM.
the use of a well-ventilated fume hood). Finally, 1.9 g of the pure hapten HOM was obtained, with recovery of product being 48% (it was calculated from m-cresol). Spectra of the hapten HOM were analyzed as follows: IR: 3 287 (s, N-H), 3 047 (m, Ar-H), 2 813-2942 (s-m, C-H), 2770-2500 (w, 0-H), 1704 (s, G O ) , 1544 (s, d&, 1488, 1440 (m, dCH,), 1237 (s, Ar-0-C), 1075 (m, C-0, carboxyl acid). In addition, only one small peak was obtained between 700 and 800 cm-I, which indicates that two sites (I-th and 3-th) on the aromqtic ring of HOM were replaced. EI: 43 (-CONH- or -NHCH,CH,-), 45 (-COOH), 56 (-CH,CH,OC-), 60 (-OOCNH-), 77 (C,H,+H+), 91 [-C,H,(CH,)], 107 (CH,C,H,O-), 121 (-C,H,OOC+ H+), 206 [C,H,(CH,) COONHC%CH,CO-], 223 (HOM'). 'H-NMR (DMSO): 6 2.35 (s, 3H, ArCH, ), 2.46-2.55 (t, 2H, HOOC-CH,), 3.50-3.61 (p, 2H, N-CH,), 5.66 (s, IH, N-H), 6.647.28 (p, 4H, Ar-H).
Preparationof hapten-proteinconjugates The hapten HOM was conjugated with proteins (BSA and OVA) using the activated ester method (AE method). One micromole of the hapten was incubated overnight with stimng at room temperature with stoichiometric amounts of N-hydroxysuccinimide and dicyclohexylcarbodiimide in 1500 pL of DMF. The mixture was cooled to 5°C for 2 h. After centrifugation, 550 pL of the clear supernatant containing the active BSA ester was added slowly to 2.5 mL, of a 12 mg d-' solution in 0.2 mol L-' phosphate buffer, pH 8.0 (another
Q2007,CMS. All rights resewed. Publishedby Elsevier Lid.
Production and Characterization of Polyclonal Antibodv for the N-Methylcarbamatc Insecticide Mclolcarb
550 yL of the clear supernatant was added to 2.5 mL of a 12 mg mL-’ OVA solution). These two mixtures were allowed to react at room temperature for 4 h with stirring, and finally the conjugates were pullfied by dialyzing for 60 h using 0.2 mol L-’ phosphate buffer, pH 6.8. Conjugate formation was confirmed spectrophotometrically. UV-visible spectra showed qualitative differences between the carrier proteins and conjugates in the region of maximum absorbance of the hapten (256 nm). The hapten to protein molar ratio of conjugates was then estimated from the spectral data of the hapten, the protein, and the corresponding conjugate.
Immunization Two female New Zealand white rabbits were used for raising polyclonal antibodies. One milligram (protein equivalent) of the immunogen (HOM-BSA) was thoroughly emulsified with an equal volume of Freund’s adjuvant. Freund’s complete adjuvant was used for the first injection, and Freund’s incomplete adjuvant was used for the subsequent booster injections. Booster injections were given at 3-week intervals. On the 7th day after each booster injection, 5 mL blood samples were taken from an ear vein to check the titer of the antiserum. The blood samples were allowed to coagulate for 2 h at 37°C and then kept in a refrigerator overnight. The serum was centrifuged at 4°C for 10 min, and the supernatant was dispensed into 1.5-mL eppendorf tubes and stored at -20°C. Booster injections were administered six times. Finally, two antisera for metolcarb, named pAbOl and pAb02, were obtained from the rabbits’ heart blood.
1 I1
per well) was added and incubated for 1 h at 37°C. The plates were washed again, and 50 yL per well of TMB solution (3.3 pL of 30% H,O,, 400 yL of 0.6% TMR in DMSO per 25 mL of acetate buffer) was added. The color development was stopped after 10-15 min by the addition of 2 mol L H,SO, (25 pL per well). The absorbance was measured at 450 nm.
Antiserum characterization Affinity and specificity are two most important characteristics that determine antiserum quality and are expressed by the titer of an antiserum and the cross-reactivity separately. Affinity: The affinity of the antiserum collected from two rabbits was detected by incompetitive ELISA, and expressed the titer was expressed as the multiple of dilution. The higher the titer, the stronger is the affinity. Therefore, the antiserum with higher titer was chosen for further research. Specificity: The cross-reactivity not only determines the specificity of the antibody but also the reliability of the assay. Data were obtained from standard curves of metolcarb, N-methylcarbamates dimethacarb, propoxur, fenobucarb, isoprocarb, carbofuran, pirimicarb, and methomyl. Each compound was prepared in 5 % methanol in PBST and tested in the concentration range 1-1000000 ng mL-’. The cross-reactivities (CR) were calculated as a ratio of the IC,,, of metolcarb to that of the tested compound. CR was set at 100% for metolcarb.
RESULTS AND DISCUSSION
Enzyme-linked immunosorbent assay (ELISA) Microplates were coated overnight at 4°C with 50 pL per well of the appropriate coating antigen concentration in carbonate-bicarbonate buffer. After the coated plates were washed with PBST, the surface of the wells was blocked with 100 yL of 1% glutin in PBS (or 1% OVA, 3% skimmed milk powder) for 1 h at 37°C. After one step of washing, 25 pL of antiserum diluted in PBS and 25 pL of analyte solution were added per well and incubated for 1 h at 37°C. After another step of washing, goat antirabbit HRP (1:2000 in PBST, 50 pL
Synthesis of hapten and its conjugation to carrier proteins A hapten mimicking the target analyte and containing reactive groups for conjugation to carrier proteins must be designed. In this article, the hapten synthesis was aimed at preserving the metolcarb structure for electronic and hydrophobic properties and considering the ideal spacer position to elicit antibodies for metolcarb. On the basis of this idea, the hapten of metolcarb (HOM) was synthesized. The synthesis of HOM was carried
02007. CAAS Ail rights reserved Published by EisevierLtd.
ZHANG Qi et al.
17X
out as shown schematically in Fig. 1. The hapten HOM with a carboxyl terminal in its structure was conjugated to carrier proteins using the active ester method. In the ultraviolet-visible spectra obtained from continuous wavelength scanning, there were several obvious changes between the spectra of the conjugate and its carrier protein. By assuming that the molar absorptivity of haptens was the same for the free and conjugated forms, the apparent molar ratios were estimated as 23 and 17 for proteins BSA and OVA, respectively (the spectral data were obtained at 256 nm).
Antiserum characterization Affinity: The antiserum titers of terminal bleeds from two rabbits (antiserum pAbOl and pAb02) were measured under identical conditions and using identical analytical plates. The results showed that both immunized individuals could produce high titer antibody, that is to say, the antibodies we produced had quite strong affinity. The titer of pAbOl is 1.28x lo6higher than that of pAbO2 (Fig.2). To identify systems that yielded the highest sensitivity for metolcarb, competitive inhibition experiments were conducted and lowest IC,, values were chosen as the optimal combinations. Results showed that the IC,, values of the AbOl and pAbO2 systems were 281.22 and 468.54 ng mL-', respectively. Therefore, the antiserum pAbOl that was not purified by IgG protein was used in this study to test the specificity of antiserum. Specificity (cross-reactivities, CR): The antiserum pAbOl showed only few CR with dimethacarb (28.2%). Because the molecular structure of dimethacarb and -c
-&..-
pAb02 pAb0l
10
2
08 06
3 '0
- 1
-
-
5
' - 6
2 3 4
.
7
___ 8 ..
20 0
0
I
10
102
103
lo*
105
106
Concentration ofmetolcarh (ng mL ')
Fig. 3 Specificity of the optimized ELISA for metolcarb. Cross reactivity values, calculated as (Isn of metolcaMI,,, of other compound) x 100, were as follows: ( I ) metolcarb, 100.0%; (2) dimethacarb, 28.2%; (3) propoxur, 0.0%; (4) fenobucarb, 0.0%; ( 5 ) isoprocarb, 0.0%; (6) carbofuran, 0.0%; (7) pirimicarb, 0.0%; (8) methomyl, 0.0%.
metolcarb is very similar, the molecule of dimethacarb had one more methyl group on the aromatic ring compared with that of metolcarb. However, for the other six N-methylcarbamate pesticides such as propoxur, the cross-reactivity rate of this method is below 0.1% (Fig.3), that is to say, there is no cross-reactivity between these pesticides and pAbO1, indicating that the antiserum pAbOl has a high specificity to metolcarb.
CONCLUSIONS Metolcarb has been widely used for controlling pests in many crops. ELISA methods that were used for rapid detection and onsite monitoring of pesticide residue are remarkable because of their several advantages. In this study, polyclonal antibody for metolcarb with strong affinity and high specificity was produced. The results showed that if ELISA conditions (such as pH value and ionic strength) are optimized, the antibody pAbOl can be used to develop a sensitive and a rapid detection protocol for metolcarb residue.
9 Q 0
04
Acknowledgements
02
0
I
4
16
64
256
512
Dilution ol' serum ( x I0 000 times)
Fig. 2 Titres of antisera against HOM-BSA.
1024
This study was supported by the National High Technology Research and Development Program (863 Program), China (2006aalOz447) and Important Technology in Agricultural Frame Adjusting of Ministry of Agriculture, China (2003-07-02A).
02007,CAAS. All rights reserved. Publishedby Elsevier Ltd.
Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb
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Oxx)7, CAAS. All rightsresewed. Publishedby ElsevierLtd.