Determination of N-Methylcarbamate Insecticide Metolcarb by Enzyme
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CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 34, Issue 2, February 2006 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2006, 34(2), 178−182. RESEARCH PAPER
Determination of N-Methylcarbamate Insecticide Metolcarb by Enzyme-Linked Immunosorbent Assay Zhang Qi1, Li Tiejun1, Zhu Xiaoxia1, Xu Lina1, Liu Fengquan1,*, Hu Baishi1, Jiang Yinghua1, Cao Bin2 1Key Lab of Monitoring and Management of Plant Diseases and Pests, Ministry of Agriculture, Department of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China 2Jiangsu Pesticide Research Institute, Nanjing 210036, China
Abstract: The hapten of the N-methylcarbamate insecticide metolcarb, 3-{[1-(3-(methyl)phenyloxy)carbonyl]amino}propanoic acid (HOM), was synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA), respectively, by the active ester method. The conjugate of HOM-BSA was used as immunogen, and we prepared rabbits' anti-metolcarb serum, the titres of which were determined by noncompetitive indirect ELISA procedures and reached 1.28×106. The results of the cross reaction indicate that the antiserum produced could specially recognize the insecticide metolcarb. After optimization of the ELISA conditions, such as ionic strengths, organic solvents, pH values, blocking agents, and so on, the proper parameters of ELISA procedure were determined, and an assay protolcol for the N-methylcarbamate insecticide metolcarb was established. On the basis of statistical analysis, the linear range of determination was from 1 to 104 ng/mL, IC50 was 40.74 ng/mL, limit detection was 0.08–0.10 ng/mL, intra-assay coefficient of variation (CV) reached 2.9%, and inter-assay CV reached 4.6%. The recoveries obtained by standard metolcarb addition to different samples such as rice, water, and soil were 80, 93.4, and 107%, respectively. This is the first time that polyclonal antibodies to the N-methylcarbamate insecticide metolcarb are produced, and the ELISA, used here for efficient and available detection of metolcarb, may become a convenient and satisfied analytical tool for monitoring metolcarb residues in environmental and agricultural samples.
Key Words: N-Methylcarbamate insecticide; Metolcarb; Hapten; Antigen; Antibody; Enzyme-linked immunosorbent assay
1 Introduction Recently, immune methods, which have been developed to detect small molecular compounds, make it possible to Structure formula of metolcarb and its hapten metolcarb, establish a new protocol for detection of metolcarb. An 3-methylphenyl methylcarbamate, is one of the N-methylcarbamate immunoassay would provide a fast, simple, and sensitive insecticides. Current analytical methods for detection of method, and can especially meet many samples detection and metolcarb are mainly conventional instruments, such as gas on-site monitoring[1, 2]. Today, there are immunoassay kits for chromatography (GC) and high-pressure liquid pesticides as commodity on sale overseas, and immunoassay chromatography (HPLC). These methods, however, have to protocols have been recognized in every country. Lots of rapid involve multistep sample cleanup procedures, and consume a immunoassays for N-methylcarbamate insecticides have been great deal of money, manpower, time, and organic solvents. reported, such as aldicarb[3], carbofuran[4,5], carbaryl[6−8], The current methods can be used only in laboratories, and methiocarb[9], and propoxur reported by Moreno et al[10]. But, people who use the instruments need to be trained specially. so far, it is not reported for production of metolcarb antigen
* Corresponding author. Email: [email protected] This work was supported by the National '863' High-Tech Research Program (2001AA246031) and the Important Technology in Agricultural Frame Adjusting (2003-07-02A), Ministry of Agriculture, China.
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ZHANG Qi et al. / Chinese Journal of Analytical Chemistry, 2006, 34(2): 178–182 and antibody. Here, we synthesized a hapten of metolcarb and bath for 2 h. After the mixture was purified and dried, 1.871 g its antigens for the first time, and prepared high-affinity and of pure HOM was obtained (yield: 48%). high-specificity antiserum for metolcarb. This hapten of HOM was verified by infrared spectra, mass spectra (EI and ESI), and NMR spectra. 2 Experimental 2.3 Synthesis and identification of the conjugates 2.1 Instruments and chemicals Antigens were synthesized using Active Ester (AE) Various instruments and chemical used in this experiment method[8]. The hapten of HOM, which has lots of terminal are as follows: UV-vis spectrophotometer-640 (Beckman, carboxyl, was conjugated to protein BSA and OVA, USA); Tensor-27 Infrared-spectrophotometer, Esquire-LC00075 respectively. Dialysis was involved to purify the conjugations. LC-ESI spectrometer, General Electric ACF-300MHz UV-vis spectral data supported the structures of the final spectrometer (Bruker, Germany); DNM-9602 microplate conjugates. The hapten density (the number of the hapten reader and DNX-9620 well-wash-plus (Prolong New molecules per molecule of protein) of conjugates was Technology Co., Beijing); standards of metolcarb and other estimated directly by mole absorbance ε.
N-methylcarbamate insecticides (≥90%, supplied by Jiangsu Hapten density = εεεconjugation− protein hapten Pesticide Research Center); bovine serum albumin (BSA), ovalbumin (OVA), and Freund′s complete and incomplete 2.4 Production and characterization of antibody adjuvant (Sigma Chemical, USA); 3,3′,5,5′-tetramethylbenzidine (TMB) and goat anti-rabbit immunoglobulin conjugated to 2.4.1 Immunization and antiserum preparation horseradish peroxidase (IgG-HRP, Huamei Biotechnology Co., China.); 96-well microplated (Corning, USA); other reagents The conjugate HOM-BSA were used as immunogen and are analytical reagents. two Female New Zealand white rabbits weighing 2.5 kg were used for raising polyclonal antibodies by the conventional 2.2 Hapten synthesis and verification protocol. On the seventh day after six times boost, two antiserums for metolcarb, named Ab01 and Ab02, respectively, 2.2.1 Synthesis of m-methylphenyl chloroformate were obtained from the rabbits′ hearts.
Activated carbon (0.1g) was added to trichloromethyl 2.4.2 Enzyme-linked immunosorbent assay (ELISA) carbonochloridate (28 g), and the reaction mixture was incubated at 36°C in water bath. Product of phosgene was Microplates were coated overnight at 4°C with 50 μL per collected by toluene (51.5 g). Till trichloromethyl well of the appropriate coating antigen concentration in 0.05 carbonochloridate was not seen again, 24% of phosgene mol/L carbonate-bicarbonate buffer (pH 9.6). After washing solution in toluene was obtained. m-Cresol (7.7 g) was added with PBST (PBS with Tween-20: 8 g/L NaCl, 1.15 g/L to 100 mL of 10% sodium hydroxide in water. Thereafter, a Na2HPO4, 0.2 g/L KH2PO4, 0.2 g/L KCl, and 0.05% slight excess of phosgene was slowly added, and the reaction Tween-20, v/v), the surface of the wells were blocked with was incubated at room temperature for 4 h with magnetic 100 μL of 1% glutin in PBS (or OVA 1%, 3% skimmed milk stirring. The organic phase was evaporated to dryness at powder) for 1 h at 37°C. After another washing step, 25 μL reduced pressure. The resulting brown solid was assumed to per well of antiserum diluted in PBS and 50 μL per well of be m-methylphenyl chloroformate (9.012 g) and subsequently analyte solution were added, and incubated for 1 h. After used without further purification. washing, IgG-HRP (1:2000 in PBST, 50 μL per well) was added and incubated for 1 h at 37°C. The plates were washed 2.2.2 Synthesis of 3-{[1-(3-(methyl)phenyloxy) carbonyl] again, and 50 μL per well of TMB solution (3.3 μL of 30% amino} propanoic acid (HOM) H2O2, 400 μL of 0.6% TMB in DMSO per 25 mL of acetate buffer, pH 5.5) was added. The color development was
The crude chloroformate (3.275 g, 12.1 mmol) was stopped after 10−15 min with 2 mol/L H2SO4 (25 μL per well). dissolved in 4 mL of cold 4 mol/L NaOH to obtain solution A. The absorbance was measured at 450 nm with a DNM-9602 β-Alanine (1.989 g, 22.3 mmol) was dissolved in 4 mL of 4 microplate reader. mol/L NaOH, and the solution was cooled at 4°C to obtain solution B. Solution A, along with 3 mL of cold 4 mol/L 2.4.3 Affinity and specificity of the antiserum sodium hydroxide (solution C), was added to solution B in five equal portions, with an interval of at least 5−10 min Affinity and specificity are two most important standards between additions. The reaction mixture was stirred in an ice for the antiserum quality, which are expressed by the titer of
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ZHANG Qi et al. / Chinese Journal of Analytical Chemistry, 2006, 34(2): 178–182 an antiserum and the cross reactivity separately. The affinity mathematical simulation equation and linear detection range, of the antiserum collected from two rabbits was detected by and evaluate the precision (intra-assay variability and noncompetitive. ELISA procedure and the titer were inter-assay variability) and sensitivity. The accuracy was expressed by the multiple of antiserum dilution. The higher evaluated by spiked samples experiment. To study spike the titer, the stronger the affinity is. So the antiserum with recovery, rice, soil, and water were spiked with different higher titer was chosen for further research. The cross concentrations of metolcarb and analyzed in a blind fashion by reactivity not only represents the antibody’s specificity, but the ELISA protocol. also decides the assay reliability. The cross reactivity is expressed by cross-reaction rate. Under the same circumstance, 3 Results and discussion indirect competitive ELISA curves of metolcarb and other analog standard were established to determine their respective 3.1 Verification of hapten
IC50 values. Cross reactivity was calculated as the ratio of
IC50: Spectra of the hapten HOM were analyzed as follows: IR:
Crossreactivity(%) = (IC50 of metocarb/IC50 of analog)×100% 3287 (s, νN−H), 3047 (m, νAr−H), 2813−2942 (s−m, νC−H), The lower the cross-reaction rate, the more specific is the 2770−2500 (w, νO−H), 1704 (s, νC=O), 1544 (s, δN−H), antiserum for metolcarb. 1488−1440 (m, δCH2), 1237 (s, νph, C−O), 1075 (m, νC−O, carboxyl acid). In addition, there is only one small peak 2.5 Competitive ELISA optimization between 700 and 800 cm−1, which indicates that two sites (1-th and 3-th) on aromatic ring of HOM were replaced. ESI: + + + Concentration of the antigen and antibody: By 224.3 (HOM+H ), 471.2 [(HOM)2+Na ], 222.6 (HOM−H ), + 1 −6 incompetitive ELISA procedure, antibodies and antigens were 445.8 [(HOM)2−H ]. H NMR [δ(×10 )]: 2.35 (s, 3H, ArCH3), screened in a two-dimensional titration for better dilution of 2.46−2.55 (t, 2H, HOOC−CH2), 3.50−3.61 (p, 2H, N−CH2), coating antigen and antiserum. The dilution multiple is 5.66 (s, 1H, N−H), 6.64−7.28 (p, 4H, Ar−H). selected as operation concentration, when OD value reaches 1.0. 3.2 The coupled identification of artificial antigen Several factors, which may have effect on ELISA curves, were determined respectively. In the UV-vis spectra obtained from continuous wavelength Ionic strength: In competitive ELISA procedure, in the scanning, there are a lot of obvious changes between the reaction system between antigen and antibody, the spectra of the conjugate and that of its carrier protein. The concentrations of NaCl were respectively 0, 0.2, 0.4, 0.8, 1.6, conjugate of uptaking peak shape is the result of lapping 3.2, and 4.0 mol/L. between the pristine carrier protein and hapten that have been Organic solvent: Three general organic solvents were tested: coupled. The result of molar ratio (hapten: protein), which was methanol, DMF, and DMSO. The contents of organic solvent assessed by HOM-OVA and HOM-BSA spectrophotometric in the reaction system were 0, 2.5%, 10%, 20% and 40%. method, is 17 and 23, respectively. Through noncompetitive ELISA, minimum effective organic solvent on the antigen-antibody reaction was chosen. After 3.3 Affinity and specificity of antiserum then, by competitive ELISA, the effect of organic solvent content toward detection results is researched. The pH values Antiserum Ab01 and Ab02 of titer were measured in same of 5, 6, 7, 8 and 9 were tested by competitive ELISA to conditions and an identical analytical plate. The results show evaluate pH effects on ELISA curves. that both the immunized individuals can bring about high titer Blocking reagent: The effect of blocking is to eliminate the antibody. The titer of Ab02 is 1.28×106 times higher than that nonspecific absorption. In this research, other conditions were of Ab01. The antiserum Ab02 was used to test its specificity. the same, while we chose 1% glutin, 1% OVA, and 3% In this study, we chose seven analogues of metolcarb, such as defatted milk power as blocking reagent (100 μL per well), dimethacarb, measured IC50 respectively, and calculated their and compared the detection curve. cross reactivity of different compounds (cross reactivity of metolcarb refers to 100%, table 1). The results show that this 2.6 Establishment of ELISA protocol and its evaluation analytical method (ELISA) can bring about cross reaction only between dimethacarb and Ab02, and the cross reactivity According to the results from section 2.5, the best operation is 28.2%, because the molecule structural of dimethacarb and parameters for metolcarb ELISA were determined, and then metolcarb is very similar (the molecule of dimethacarb under the above-mentioned conditions, competitive inhibition consists of one methyl more on the aromatic ring than that on ELISA in series concentrations of metolcarb standard was the metolcarb molecule). However, for the other six repeated five times at different times in order to work out the N-methylcarbamate pesticides, such as propoxur, the cross
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reactivity of this method is below 0.1%. This indicates that the IC50 (0.8 mol/L) < IC50 (0.4 mol/L) < IC50 (0.2 mol/L) < IC50 antiserum produced can be used to develop an ELISA protocol (0.1 mol/L). Fig. 1b reveals that DMF has an effect on the which has high specificity to metolcarb. combination of antibody and antigen, and methanol is least affected. So methanol was selected and the effect of its Table 1 Cross reaction of antibody Ab02 with metolcarb and its various contents was studied further by indirect competitive
analogues ELISA; the result shows that the sequence of IC50 is methanol Cross Cross 20%, 10%, 5% (Fig. 1c). The effect of various pH was
IC50 reaction IC50 reaction obtained by competitive ELISA (Fig. 1d). If the matrix is Analogues Analogues (ng/mL) rate (ng/mL) rate acidic, the cross reaction between antiserum and blocking (%) (%) OVA will be heavy. And the cross reaction depressed the Metolcarb 512.86 100 Isoprocarb >1,000,000 <0.1 ability of antibody to combine with metolcarb. When pH Dimethacarb 1,819.70 28.2 Carbfuran >1,000,000 <0.1 equals 8 and 9, there is no obvious difference in the detection
Propoxur >1,000,000 <0.1 Pirimicarb >1,000,000 <0.1 curve, and IC50 is relatively low. We compared the effect of Fenobucarb >1,000,000 <0.1 Methmyl >1,000,000 <0.1 three conventional blocking agents and found that using 1% gelatin as blocking agent will be most beneficial in 3.4 Competitive ELISA optimization establishing a high-sensitive ELISA for metolcarb. Through studies of several factors, the main parameters of Concentration of antibody and antigen: The concentrations ELISA procedure were determined: concentration of coating of antibody and coating antigen were best determined by the antigen HOM-OVA was 0.6 μg/mL, dilution of Ab02 was two-dimensional titration method. The dilute concentration of 1/2000, the blocking reagent was 1% gelatin, the co-solvent coating antigen HOM-OVA is 1:10,000, which equals 0.6 was 5% methanol, pH was 7.4−9, and ionic strength was 3.2 μg/mL of protein and the dilute concentration of antiserum mol/L. Ab02 is 1:20,000. Fig. 1a represents the consequence at
various ionic strengths: IC50 (3.2 mol/L) < IC50 (1.6 mol/L) <
Fig. 1 Effect of ionic strengths (a), kinds (b), concentration (c) of organic solvent, pH (d) and blocking agents (e) on ELISA curves
3.5 Establishment and evaluation of ELISA protocol concentration of metolcarb (ng/ml) as the longitudinal coordinates (x). After conversion of Fig. 2a, we could observe 3.5.1 Establishment of ELISA and evaluation of precision that in the range of 1−10,000 ng/mL, the graph between
and sensitivity logit(B/B0) and logarithm of concentration of metalcarb (ng/mL) was linear (Fig. 2b), and the regression equation was Under the optimized conditions mentioned previously, the obtained (y = 0.9404x+1.5146, R2 = 0.9939). In this optimized
indirect competitive ELISA procedures were conducted in ELISA, IC50 value was 40.74 ng/mL and the LD (limit quintuplicate with a series of standard concentration of detection) was 0.08−0.10 ng/mL by the extrapolation of
metolcarb at different times. Then a competitive curve B0-3SD extrapolation. representing the average was obtained (Fig. 2a). We used logit The variability of intra-assay and inter-assay of the ELISA
(B/B0) as the lateral coordinates (y), logarithm of curve for metolcarb was used to show the precision of this
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ZHANG Qi et al. / Chinese Journal of Analytical Chemistry, 2006, 34(2): 178–182 protocol. The intra-assay variability was given by the average stable in different microplates. of six replicated wells in one microplate. The inter-assay Table 2 The variability of intra-assay and inter-assay of the ELISA variability was given by the average of five replicated curve for metolcarb microplates at different times. These calculation methods were Concentration of Intra-assay mean Meter-assay mean [11] based on reference . metolcarb (µg/L) binding±SD binding±SD (RSD%) On the basis of Table 2, the intra-assay average variation 1 73.5±3.5 80.5±6.1 (7.6) coefficient was 2.9%, which indicated that measured values 10 55.5±2.3 64.9±6.8 (10.5) from identical microplate could be repeated highly. In the five 100 39.6±2.0 43.6±30 (6.8) duplicated microplate assays, the average variation coefficient 1000 18.0±1.2 23.0±3.9 (16.8) of the calibration curve in different concentrations was in the 1000 6.60±1.9 8.40±1.3 (15.2) range of 6.8%−16.8%, and the average was 4.6% (Table 2), Mean 38.6±2.2 44.1±4.2 (11.4) which suggested that the established ELISA method was
Fig. 2 Indirect competitive ELISA curve for metolcarb
3.5.2 Spiked Recovery tests (Evaluation of accuracy) Table 3 Recovery test of metolcarb in water, soil and rice Found Mean recovery Sample Theoretical (µg/L) The spiked recovery mean and sanity were used to represent (%) ±SD (%) the accuracy of this indirect competitive ELISA. 225.7 (1) Spiked Recovery: Tap water, soil and rice were spiked 213.4 200 104.6±6.6 with a certain amount of metolcarb standard which were 196.1 analyzed by the ELISA after dilution to a certain extent (the 201.6 soil and the rice samples had been soaked in water for about 2 52.8 h first). According to the B/B0 (or OD) values we found the 56.0 50 104.3±6.0 corresponding concentration of metolcarb from the calibration 49.2 curves. After the assay, the spiked recovery mean in water by 50.0 Water the established ELISA method for metolcarb was 98%−120%, 10.2 107% on average; spiked in soil, the recovery mean was 11.6 10 109.5±10.7 68%−88%, 80% on average (Table 3). 9.9 (2) Sanity: The curves established by the water and soil 12.1 samples spiked with metolcarb in three concentrations 391.1 respectively were compared. The results demonstrated the 436.7 500 82.2±3.8 curves of water and soil samples were parallel to the 403.3 calibration curves basically (data in Table 3, figure omitted). 412.5 101.4 4 Conclusions 112.7 125 81.8±6.4 101.6 Immunoassay is a sensitive, rapid and practical method and 93.2 Soil has a broad applied perspective for the detection of pesticide 16.8 residues. In our study, it was originated of the ELISA method 18.2 25 77.0±9.1 established for the detection of metolcarb. Microplates coated 22.1 and blocked could be reserved at least for 2 weeks at 4°C. It 19.9 only took 2.5 h to measure the analyte residue using the 90.6 microplates which were blocked before, so the method is 94.2 Rice 100 95.4±7.4 suitable for the rapid detection of metolcarb residues in water, 103.2 soil, rice, or the farming products, and so on. 85.7
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