Environ. Sci. Technol. 2007, 41, 6783-6788
Enzyme-Linked Immunosorbent Assay Based on a Monoclonal Antibody for the Detection of the Insecticide Triazophos: Assay Optimization and Application to Environmental Samples
In a previous paper (12), a very sensitive ELISA for triazophos based on the THHe monoclonal antibody and heterologous THBu-HRP conjugate has been developed (I50 ) 0.65 µg/L; I20 ) 0.10 µg/L) .The present work describes the influence of several physicochemical factors on its performance and the ELISA optimization process. Likewise, several organophosphorus insecticides or analogues as competitors were tested to evaluate the specificity of the assay. The optimized ELISA was finally applied to the determination of water and soil samples spiked with triazophos to test the feasibility of the ELISA.
CHIZHOU LIANG, RENYAO JIN, WENJUN GUI, AND GUONIAN ZHU* Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou, 310029, China
Experimental Section
A direct competitive enzyme-linked immunosorbent assay (ELISA) for triazophos was developed, which was based on the THHe monoclonal antibody (McAb) and a heterologous enzyme tracer (THBu-HRP). The influence of several physicochemical factors (temperature, time, pH, salt, detergent, and solvent) on the immunoassay was studied. For the standard curve, an I50 of 0.21µg/L and a limit of detection (I20) of 0.02µg/L was obtained in a high salt concentration buffer (0.05 M PBS, pH 6.0) with 0.05% BSA, which means an almost 3-fold improvement in the assay sensitivity in comparison with the nonoptimized conditions. The optimized ELISA has been used to quantify triazophos in water and soil samples spiked at different amounts. The excellent recoveries achieved confirmed the potential of the immunoassay for environmental monitoring of triazophos in waters and soils without purification steps.
Introduction Triazophos [O, O-diethyl O-(1-phenyl-1H-1, 2, 4-triazol-3yl) phosphorothioate], an organophosphorus insecticide, is a broad-spectrum insecticide and mainly applied to paddy rice pest control in China. In recent years, most high-toxic and high-residual organophosphorus pesticides, for example, methamidophos, parathion, and methyl parathion, were banned for use on crops by the Agriculture Adaministration of China. As a good alternative, triazophos has more widespread applications. Triazophos is stable and degrades relative slowly in the environment (1-3). Hazard and potential risk to human and nontarget species of its residue in food and environment is a growing concern. Analytical methods involving gas chromatography and liquid chromatography have been used successfully for the detection of triazophos residues (3, 4-8). But chromatographic methods are laborious and time-consuming and require sophisticated equipment available in only wellequipped laboratories. ELISA is proven to be simple, costeffective, and do not require sophisticated instrumentation (9). Furthermore, the immunoassay is demonstrated as a powerful tool involved in high sample throughput and onsite screening in pesticide monitoring programs (10, 11). All of these features make the immunoassay a very promising analytical tool. * Corresponding author e-mail:
[email protected]. 10.1021/es070828m CCC: $37.00 Published on Web 09/05/2007
2007 American Chemical Society
Immunoreagents, Chemicals, and Instruments. The production of the THHe McAb and the preparation of the THBuHRP assay conjugate have been described in a previous paper (12). Figure 1 shows the structures of the immunogenic hapten [THHe, O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(5-carboxyamyl) phosphoramidomidothioate] and assay hapten [THBu, O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) N-(3-carboxypropyl) phosphoramidothioate]. Triazophos structure is also presented for comparison. Triazophos and other pesticide standards were obtained from National Standards Company (China). Horseradish peroxidase (HRP), ovalbumin (OVA, MW45000), and bovine serum albumin (BSA, MW 67000) were obtained from Sigma (Spain). Ophenylenediamine (OPD) and Tween 20 were purchased from Shanghai Chemical Reagents Company (China). All other chemicals and organic solvents were of analytical grade or better. The ELISA was carried out in 96-well polystyrene microplates (COSTAR, High Binding Plates, U.S.). Plates were washed with a DEM plate washer (Beijing Tuopu Analytical Instruments Co. Ltd., China) and absorbencies were read with a 550 plate reader (Bio-Rad, America). ELISA Protocol. The immunoassay to be optimized was a direct ELISA based on the coating THHe McAb and the heterologous enzyme tracer (THBu-HRP). All incubations were carried out at 37 °C. Standards were prepared in PBS1 (PBS containing 274 mM NaCl, pH 7.4) and methanol (MeOH) (PBS1:MeOH, 9:1, v/v) by serial dilutions from a stock solution in methanol using borosilicate glass tubes. The ELISA was run as described in a preceding paper (12). Briefly, 96-well microplates were coated with the antibody (100 µL/well, 8 µg/mL in 0.01 mol/L PBS, pH 7.4) by incubation for 2 h. Plates were washed four times with PBST (PBS containing 0.05% Tween 20, pH 7.4). Binding sites not occupied by the coating antibody were blocked with 2% of skimmed milk in PBS (300 µL/well) by incubation for 30 min. Plates were then washed as before. 50 µL/well of triazophos standards or samples were added in quadruplicate, followed by 50 µL/ well of THBu-HRP (0.4 µg/mL, in 4% skimmed milk-PBS1). After incubation for 1 h, the plates were washed again and 100 µL/mL of OPD solution (10 mg of OPD and 10 µL of 30% of H2O2 diluted with 25 mL of phosphate-citrate buffer, pH 5.4) was added. After incubation for 15 min, the reaction was stopped by adding 50 µL/well of 2 M sulfuric acid, and absorbance at 490 nm was read and recorded. Effect of Incubation Buffer Concentration. The effect of buffering capacity of assay solution on ELISA performance was studied using different concentrations of buffers to dissolve hapten-HRP and in 10% MeOH-water to dissolve triazophos standards. The buffers were prepared by diluting 20 × PBS (10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) with distilled water. The pH of all buffers was VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Soil Properties sample yellow-red soil purple clayed paddy soil silt-loamy paddy soil coastal saline soil
pHa OMb (H2O) (%) 5.0 5.8 5.4 8.3
1.0 5.2 0.9 0.8
silt (%)
sand (%)
clay (%)
CECc (meq 100 g-1)
23.1 42.1 44.6 30.1
15.7 11.5 46.3 56.0
61.2 46.4 9.3 13.9
8.8 18.2 21.0 6.7
a pH values were obtained by measuring the solutions of soil in water (soil:water, 1:2.5, g/g). b OM (organic matter); c CEC (cation exchangeable capacity).
FIGURE 1. Chemical structures of triazophos and the haptens used in the immunoassay. kept between 7.3 and 7.5. The competitive curves were performed with above hapten-HRP and triazophos standards prepared. pH Effect. The influence of pH of assay solution on immunoassay performance was studied using buffers of various pH values to dissolve hapten-HRP and in 10% MeOHwater to dissolve triazophos standards. The buffers were 100 mM PBS at different pH values, which were prepared by changing the amounts of Na2HPO4 and KH2PO4, whereas the concentration of NaCl and KCl remaining at 1.37 M and 27 mM. The competitive curves were performed with above hapten-HRP and triazophos standards prepared. Tween 20 Concentration Studies. To study the effect of Tween 20 on immunoassay performance, the hapten-HRP was dissolved in the concentrated PBS (cPBS: 50 mM sodium phosphate, 685 mM NaCl, 13.5 mM KCl, pH 6.0) containing different concentrations of Tween 20 (from 0 to 1.0%, v/v) and the triazophos standards were dissolved in 10% MeOHcPBS. Then the hapten-HRP and triazophos standards prepared above were used to perform competitive curves. Solvent Effect. The assays’ tolerance to methanol, acetonitrile and acetone was evaluated between 0 and 20% solvent concentration (v/v). In this case, competitive curves were performed from the triazophos standards dissolved in cPBS containing different amounts of methanol, acetonitrile and acetone, and hapten-HRP in cPBSB (cPBS with 0.05% BSA). Cross-Reactivity Determinations. To evaluate the specificity of the THHe McAb, triazophos and several related compounds were tested for cross-reactivity (CR). CR was calculated using the following equation: [I50 (triazophos)/I50 (compound)] × 100%. Here, I50 value (analyte concentration that reduces 50% maximum absorbance of the competitive ELISA) was determined by performing competitive immunoassay. A competitive curve was obtained by plotting absorbance against the logarithm of analyte concentration. Sigmoid curve was simulated by means of Microsoft Excel 2000. Water and Soil Sample Analysis. The optimized ELISA was used for triazophos determination in six water samples from different sources, tap water (Hangzhou, China), pond water (Huajiachi Pond, Hangzhou, China), lake water (West Lake, Hangzhou, China), paddy water (experimental paddy field, Zhejiang University), river water (Tiesha River, Hangzhou, China), and commercial bottled water. Water samples showed a pH between 5.8 and 6.6. Prior to the ELISA determination, all samples were filtered with filter paper (φ12.5 cm) and diluted with concentrated PBS2 (cPBS2:200 mM sodium phosphate, 2.74 M NaCl, 54 mM KCl, pH 6.0) (water: cPBS2, 3:1, v/v). For ELISA analysis, 9.75 mL of water samples were spiked with 0.25 mL of triazophos standard in methanol at different concentrations covering the quantitative working range and analyzed directly without further extraction. 6784
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In addition, four soil samples from different locations (Table 1 shows the property of each soil), yellow-red soil (Quzhou, China), purple clayed paddy soil (Hangzhou, China), silt-loamy paddy soil (Hangzhou, China), coastal saline soil (Ningbo, China), were collected, dried, and passed through a 60 mesh sieve. 20.0 g soils, free of triazophos residues tested by gas-liquid chromatography, were distributed into glass jars and spiked with 400 µL of triazophos standards. The soils were mixed thoroughly with a stainless steel spatula for 5 min and then allowed to equilibrate for 24 h before being aliquoted into 10 replicates of 2.0 g samples. Methanol (4.0 mL) was added to 2.0 g of soils and vortexed for 1 min. The mixtures were then sonicated for 5 min and centrifuged for 10 min (4000 rpm). The superstratum (2.0 mL) was transferred and evaporated by gently blowing nitrogen to move off the solvent. The residue was dissolved with 10.0 mL of 2.5% MeOH-cPBS. The residue was determined by the optimized ELISA after vigorously shaking it for 5 min by a rotation shaker.
Results and Discussion ELISA Optimization. With the aim of improving immunoassay performance, the influence of several nonspecific parameters on assay characteristics was examined. I50, maximal absorbance (Amax), dynamic range (DR), and limit of detection (LOD) are usually used as criteria to evaluate immunoassay performances. Amax/I50 is a convenient estimate of the influence of some factors on the ELISA sensitivity, the higher ratio indicating higher sensitivity (15). In this work, the Amax/I50 ratio was used as the primary criteria to evaluate the immunoassay performances, and DR and LOD were also taken into account. Both temperature and time are important physical parameter for the immunoassay. In general terms, the higher the temperature was, the shorter the incubation time needed, and an increase of the incubation time resulted in higher maximum absorbance and slightly lower triazophos I50. However, at higher temperature, the intraassay coefficient of variation (CV) noticably increased . Therefore, incubation temperatures of 23-25 °C and incubation times of 2 h for the competition step and 15 min for the enzymatic reaction were selected and used throughout this work. Salt Concentration. The plots of assay parameters (Amax, I50) as a function of salt concentration were depicted in Figure 2. As shown, the ability of McAb to recognize the corresponding conjugated hapten (Amax) increased gradually as buffer salt concentration increased. In addition, the recognition of triazophos (I50) did not change markedly apart from 20 × PBS concentration as a result of salt concentration increase. The Amax/I50 ratio was used to select the optimum buffer concentration. The ratio increased gradually with the salt concentration up to 10 × PBS, then decreased. Therefore, 10 × PBS concentration was selected as the assay buffer (the actual salt concentration of the assay buffer was 5 × PBS). Identical results (13, 14) concerning the effect of the salt concentration have been previously found in similar studies with several other nonpolar pesticides, such as chlorpyrifos
FIGURE 2. Effect of the salt concentration of the assay buffer on the analytical characteristics of triazophos competitive standard curve: (9) absorbance in the absence of triazophos (Amax) and (2) value of I50 for triazophos. For the competition step, standards were prepared in distilled water and methanol (distilled water:methanol, 9:1, v/v), and the enzyme tracers were diluted in buffers of different ionic strengths which were prepared by diluting 20 × PBS with distilled water, pH 7.4, containing 4% skimmed milk. Each point represents the average of four replicates.
FIGURE 3. Effect of the pH of the assay buffer on the analytical characteristics of triazophos competitive standard curve: (9) absorbance in the absence of triazophos (Amax) and (2) value of I50 for triazophos. For the competition step, standards were prepared in distilled water and methanol (distilled water: methanol, 9:1, v/v) and the enzyme tracers were diluted in the 10 × PBS of different pH, containing 4% skimmed milk. Each point represents the average of four replicates. and carbofuran, which were determined by the AC (antibodycoated) format. Also, an improvement in maximum signal and sensitivity owing to the increase of salt concentration has been reported (15). In addition, there was a study (16) indicating that Amax and I50 decreased gradually as the buffer salt concentration increased. On the contrary, the opposite effect on both parameters was observed with the polar compound TCP, chlorpyrifos, and carbofuran, which were determined by the CC (conjugate-coated) format (13, 14, 17). All of these findings suggest that hydrophobic interactions are predominant in the nonpolar analyte-antibody binding, which are favored by increasing the ionic strength of the reaction medium, whereas the increase exerts a detrimental influence on interactions where ionic driving forces predominate. Moreover, the effect of salt concentration is perhaps related to the ELISA format, too. pH. Many studies (15, 18-20) revealed the effect of pH on both signal and sensitivity. Just like the influence of the salt concentration, the results were not all the same. The immunoassay for triazophos was pH-sensitive, as seen in Figure 3. On the one hand, maximum absorbance increased with pH to reach the maximum at pH 6 and then diminished gradually as pH increased. On the other hand, the immunoassay showed a tendency to increase the affinity to triazophos (lower I50 value) with increase of pH. Identical effect of pH on immunoassays was obtained for carbofuran (14). Considering the nonionizable nature of triazophos, the effect observed may be related to the conformational changes of the macromolecules participating in the interaction. On the basis of the result that Amax/I50 ratio was maximal at pH 6 and of the fact that triazophos was more stable in acidic and neutral environments (1), it can be seen that pH 6 seemed to be a reasonable choice for acidity of the buffer of the competition step.
Effect of Additive Tween 20, BSA, and Skimmed Milk. Detergents are commonly used in immunoassays to reduce nonspecific binding or improve sensitivity. There is a study (21) showing that the nonionic detergent Tween 20 in buffers reduced nonspecific binding and improved sensitivity. However, there are many papers (13, 15, 22, 23) proving that Tween 20 has markedly the opposite effect on ELISA performance which seems due to nonspecific hydrophobic interactions between the detergent and the nonpolar analytes, accordingly disturbing the specific analyte-antibody interaction. To investigate what effect this reagent had on the triazophos assay, the influence of Tween 20 on both the signal and sensitivity was studied, using the optimized buffer concentration and pH conditions with different Tween 20 concentrations. In our study, the negative influence was also observed as Tween 20 was included in assay buffer (higher I50 value), as seen in Figure 4. This result could be also related to the nonspecific hydrophobic interactions between the detergent and the analyte triazophos. Significantly, the positive effect of Tween 20 was obvious, which the maximum absorbance measured in the assay buffer with Tween 20 was visually higher than that without the detergent. Sometimes, BSA instead of Tween 20 included in assay buffer could obtain satisfactory result (15). Skimmed milk powder is usually added to the assay buffer to enhance the sensitivity, reduce well to well variability, or to lower background for pesticide immunoassays. Their influence on triazophos immunoassay was also studied in present study. As shown in Figure 4, BSA and skimmed milk have similar effects on triazophos assay. That is, the maximum absorbance improved as BSA and skimmed milk added in the competition buffer increased, whereas, as they increased, the sensitivity of the immunoassay slightly decreased. Considering that the McAbs in our work were prepared by immunizing mice with BSA-hapten conjugates, we originally thought that BSA would react with the McAbs. But the result indicated that the McAb of THHe did not recognize the BSA. This interesting result needs to be investigated further. Based on Amax, I50, and Amax/I50, when Tween 20, BSA, or skimmed milk was included in the assay buffer, a conclusion seemed to be drawn that the buffer with 0.05% BSA was the best. Furthermore, to take other relevant parameters, such as background absorbance (BA) and signal coefficients of variation (CV) into account, 0.05% BSA was still the best selection of them. The mean BA and CV was 0.020, 0.021, and 0.023 and 7.6%, 6.1%, and 8.5%, respectively, when 0.01% Tween 20, 0.05% BSA or 0.3% skimmed milk was added into the assay buffer. Solvent Tolerance. As a nonpolar pesticide, triazophos is commonly dissolved in solvent, and standards are prepared in PBS or water by serial dilutions from the stock solution. Moreover, solvents are often used to extract analytes from samples in the immunoassays. Therefore, it is necessary to examine what effect the solvents have on immunoassays. In our work, the effect of methanol, acetonitrile, and acetone on the ELISA performance was evaluated by preparing standard curves in buffers containing various amounts of organic solvent. As seen in Figure 5, a similar trend of the three solvents effect was observed, i.e., an improvement in maximum signal and sensitivity was obtained with increase of the solvents. The signal and sensitivity reached a maximum at a certain concentration and then decreased. Similar trend of solvent effect was found for studies of polychlorinated dibenzo-p-dioxins immunoassays (13). From the result, it may be speculated that the increasing polarity of the medium at the competition step by using lower concentration of organic solvent leads to the increase of hydrophobic antibody-analyte interactions and to the conformational changes of the macromolecules participating in the interaction which were more similar to the physical states. Interestingly, from VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Influence of Tween 20, BSA and skimmed milk of the assay buffer on the analytical characteristics of triazophos competitive standard curve: (9) absorbance in the absence of triazophos (Amax) and (2) value of I50 for triazophos. For the competition step, standards were prepared in 10% MeOH-cPBS, and the enzyme tracers were diluted in cPBS, containing different amounts of Tween 20, BSA, or skimmed milk. Each point represents the average of four replicates.
FIGURE 5. Influence of different organic solvents on the analytical parameters of the triazophos competitive standard curve in immunoassay: (9) absorbance in the absence of triazophos (Amax) and (2) value of I50 for triazophos. For the competition step, standards were prepared in cPBS containing different concentration of the solvents, and the enzyme tracers were diluted in cPBS containing 0.05% BSA. Some I50 data are not present in the graphs because of absorbance values not properly fitting to the four-parameter logistic equation. Each point represents the average of four replicates. the estimation of the Amax/I50 ratio against the solvents concentrations, the optimal concentrations of the solvents in the buffer were all around 2.5%. Similar to several other workers’ conclusion (13, 14), the methanol caused the least negative effect on the triazophos immunoassay, whereas the acetone caused the most. An unexpected advantage of using a lower amount of organic solvent was the possibility of lowering the optimal coating McAb concentration from 8 µg/L to 6 µg/L. Taking all these factors into account, the optimized conditions for the triazophos immunoassay is summarized as follows: a coating monoclonal antibody (THHe) concentration of 6 µg/L, a enzyme tracer (THBu-HRP) concentration of 200 µg/L, room temperature for all incubations, an incubation time of 2 h for the competitive step, a 15 min for the enzymatic reaction, 0.05 M PBS (pH 6.0) with 0.05% BSA for the competitive step and 2.5% methanol in assay buffer. Specificity of McAb THHe. Several compounds were tested for cross-reactivity (CR) to evaluate the specificity of the THHe McAb (Table 2). The McAb proved to be very specific for triazophos, since none of the assayed compounds was recognized (CR < 0.01%) except the hapten synthesized for this work. The result was similar to the one of previous work (12). From the result it may be speculated that the triazole ring was critical for antibody recognition as the interference of the compounds with two EtOs attaching to phosphorus atom or aromatic ring was negligible. Analysis of Water and Soil Samples. Because the ionic strength and pH of the medium was proved to influence the triazophos immunoassay, cPBS2 was added to water samples (cPBS2:water, 1:3, v/v) to make their conductivities equal to those of PBS. Then standard curves were prepared in water samples and in 0.05 M PBS as control. The assay parameters showed no noticeable difference between triazophos standard curves obtained in waters and in PBS. Consequently, it seemed applicable to perform direct determination of 6786
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triazophos in environmental waters without further treatment. Water samples from different sources were fortified with triazophos at 0.050, 0.500, and 1.500 µg/L. Each fortified sample was analyzed four times in quadruplicate by the immunoassay systems herein studied. Results were shown in Table 3. The average recovery and intraassay CV was 90 and 11.3%, ranging from 64 to 108% and from 6.8 to 18.8%, respectively. Control samples without triazophos were also systematically included in the analysis, and values were obviously lower than the assay detection limit (0.02 µg/L) in all cases, so false positives were not observed. Additionally, soil samples were also analyzed by the ELISA after being fortified with triazophos. As shown in Table 4, the recoveries were between 75 and 117% when soil samples were spiked with triazophos at 0.500, 1.000, and 2.000 µg/kg. The mean assay CV was also satisfactory, which was 11.4% in average and ranged from 6.5 to 18.7%. It needs to be pointed out that the dilution of 1:20 was insufficient for diluting out matrix interferences in the purple clayed soil. For this sample, positive results were obtained when a blank was assayed, suggesting important matrix effects. A 100-fold dilution was needed because only in this or higher dilution, the absorbance was significantly lower than that of PBS which served as control. In summary, the immunoassay can be affected by many factors and optimization process is necessary to improve the sensitivity, accuracy, and reproducibility. The application of the ELISA to the determination of tirazophos in environmental samples showed good characteristics with simple operation, high sensitivity, good specificity, and celerity, which indicated the potential of this approach to control triazophos in the environment. The results also showed dilution of the samples was an effective alternative to circumvent matrix effects when the matrix interferences affected the activity of tracer enzyme and/or antigen antibody interaction. It was worth pointing out that the phenomenon of the time-dependent drift was observed in this work. To
TABLE 2. Cross-Reactivities of the Triazophos for Related Compounds
a
Standards were diluted in cPBS and methanol (cPBS: methanol, 9.75: 0.25, v/v).
TABLE 3. Analysis of Triazophos-Spiked Environmental Waters by ELISA
sample
triazophos triazophos fortified detected recovery SDa (µg/L) (µg/L) (%) (µg/L)
CVb (%)
tap water
0.050 0.500 1.500
0.043 0.510 1.622
86 102 108
0.006 14.0 0.054 10.6 0.144 8.9
pond water
0.050 0.500 1.500
0.038 0.475 1.572
76 95 105
0.005 13.2 0.049 10.3 0.123 7.8
lake water
0.050 0.500 1.500
0.037 0.512 1.482
74 102 99
0.005 13.5 0.052 10.2 0.101 6.8
paddy water
0.050 0.500 1.500
0.032 0.425 1.328
64 85 89
0.006 18.8 0.066 15.5 0.141 10.6
river water
0.050 0.500 1.500
0.045 0.456 1.356
90 91 90
0.005 11.1 0.039 8.6 0.121 8.9
bottled water
0.050 0.500 1.500
0.045 0.402 1.456
90 80 97
0.007 15.6 0.041 10.2 0.125 8.6
90
11.3
mean
SD (standard deviation n ) 4). CV (coefficient of variation): data obtained from four determinations performed in the same ELISA plate. a
TABLE 4. Analysis of Triazophos-Spiked Environmental Soils by ELISA
b
overcome this problem, a blank 96-microplate was used. Since discrete sample containers need to be manipulated and attention must be paid to avoid cross-contamination
sample
triazophos triazophos fortified detected recovery SDa CVb (µg/kg) (µg/kg) (%) (µg/kg) (%)
yellow-red soil
0.500 1.000 2.000
0.422 0.946 2.153
84 95 108
0.07 0.13 0.16
16.6 13.7 7.4
purple clayey soil
0.500 1.000 2.000
0.374 0.788 1.892
75 79 95
0.07 0.12 0.19
18.7 15.2 10.0
silt-loamy paddy soil
0.500 1.000 2.000
0.454 1.049 2.131
90 105 107
0.06 0.11 0.14
13.2 10.5 6.5
coastal saline soil
0.500 1.000 2.000
0.423 0.904 2.340
85 90 117
0.06 0.10 0.21
14.2 11.1 9.0
mean
94
11.4
SD (standard deviation n ) 4). b CV (coefficient of variation): data obtained from four determinations performed in the same ELISA plate. a
between samples, the pipetting step tends to be the slowest step. We first transferred enough amounts of the standards and samples to the blank 96-microplate, for example, if the standards and samples were quadruplicate, 250 µL was needed. Then they were dispensed into the antibody-coated plate with 8- or 12-channel pipet. Thus, the time of pipetting step was shortened markedly and the results showed no significant drift. Furthermore, a better reproducibility between replicates was obtained. VOL. 41, NO. 19, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Acknowledgments We are very grateful to National Standards Company (China) for providing us with pesticide standards and to Zhejiang Health Creation Biotech Co. Ltd. for financing support. This work was supported by the National Natural Science Foundation of China (30370944) and Zhejiang province Key Technologies R & D Programme(2006C12102).
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(15)
(16)
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(20)
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Received for review April 9, 2007. Revised manuscript received July 9, 2007. Accepted July 23, 2007. ES070828M
