Environmental Immunochemical Methods - American Chemical Society

Chapter 17

Evaluation of an Automated Immunoassay System for Quantitative Analysis of Atrazine and Alachlor in Water Samples Downloaded by STANFORD UNIV GREEN LIBR on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch017

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Barbara Staller Young , Andrew Parsons , Christine Vampola , and Hong Wang 3

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Millipore Corporation, 80 Ashby Road, Bedford,MA01730 EnSys Inc., Royal Center, 4222 Emperor Boulevard, Morrisville,NC27560 Environmental Health Laboratories, 110 Hill Street, South Bend,IN46617 2

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Two commercially available immunoassay kits were evaluated in conjunction with an automated microplate system for the detection of atrazine and alachlor in water. The results showed that excellent precision and accuracy were achieved. Eighty six water samples could be analyzed in approximately 2.5 hours, including sample and instrument set-up time. This study supports the concept that immunoassay technology would be useful for rapid, accurate screening of water samples for the presence of various pesticides. The automated system would be particularly effective when large numbers of samples must be analyzed. Immunoassay technology has proven to be a useful tool for performing rapid, accurate, and cost-effective screening of field samples on site or in the laboratory (1-4). The goal of this study was to establish the feasibility of screening large numbers of drinking water samples for the presence of pesticides using commercially available ELISA (enzyme linked immunosorbent assay) plate kits (EnviroGard™, Millipore Corporation) in conjunction with an automated microplate system (ELs 1000, Bio-Tek Instruments, Inc.). Two EnviroGard™ kits (for triazines, and alachlor) were evaluated and found to give excellent precision and accuracy for the detection of atrazine and alachlor in fortified Milli-Q and drinking water samples. General Description of Immunoassay Kits Both the kits for triazines and alachlor are based on a standard microtiter plate competitive ELISA format. Calibrators or samples are added to the 0097-6156/96/0646-0183$15.00/0 © 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by STANFORD UNIV GREEN LIBR on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch017

184

ENVIRONMENTAL IMMUNOCHEMICAL METHODS

wells, followed by an enzyme conjugate. After a one hour incubation during which time the analyte present in the sample competes with enzyme conjugate for antibody binding sites, unbound reagents are washed away. Following the wash step, substrate is added and color is allowed to develop for 30 minutes. Color development is halted with the addition of stop solution, and the results are read at a wavelength of 450 nm. The ELs 1000 automated system is designed to perform all of these steps including data analysis. Millipore and Bio-Tek scientists worked together to design software programs tailored to the EnviroGard™ assays. Using the ELISA kits with the automated system, two full microtiter plates can be run at a time, allowing for the analysis of up to 86 water samples in approximately 2.5 hours, including set-up time. Clean-up time between runs takes less than 30 minutes.

Performance of the Alachlor Assay Table I shows the calibration curve for the alachlor plate kit. The assay is calibrated during each run by including five standards that are supplied in the kit. The dynamic range for this assay is 0.1-2.5 ppb alachlor. Typically, standards and samples are run in duplicate, and the absorbance values are converted to % B by dividing the non-zero calibrator absorbance values by the value for the 0.0 ppb calibrator. There are specified % B ranges delineated in the product inserts, serving as a quality control check for assay performance. For instance, the acceptable ranges for the alachlor product are 64-86% for the 0.1 ppb calibrator, 33-55% for the 0.5 ppb calibrator, and 11-21% for the 2.5 ppb calibrator. The precision for duplicate calibrators shown in Table I is quite good, with % coefficient of variation (%CV is defined as (standard deviation/mean) χ 100) values ranging from 0.26 to 1.56. The alachlor plate assay was found to give very reproducible results over time. In Table II, precision was determined by mnning the calibrators in duplicate on five separate days. The % B values over the 19 day period were very consistent, yielding %CVs from 1.15 to 2.53. The accuracy and precision of the assay were determined by measuring recovery of alachlor spiked into Milli-Q water at 1.0 ppb and 0.1 ppb. Table III summarizes data where triplicate samples were run on 5 separate days. The calculated %CVs for alachlor concentrations were excellent, averaging just over 7% for both spiked levels. The recovery ranges were 93-123% for 0.1 ppb alachlor, and 96-120% for 1.0 ppb. These data demonstrate excellent accuracy and precision for alachlor determinations in spiked reagent grade water samples. 0

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In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Automated System for Atrazine & Alachlor

Table I Alachlor Plate Kit


Calibration Curve on Automated System Standard Value (ppb)

Absorbance (450)

0.0 0.0 0.10 0.10 0.25 0.25 1.00 1.00 2.50 2.50

1.949 1.939 1.478 1.511 1.217 1.212 0.722 0.731 0.424 0.417

%B

Mean % B

0

76.03 77.73 62.60 62.35 37.14 37.60 21.81 21.45

% C V of Absorbance

0

100.00

0.26

76.88

1.56

62.47

0.29

37.37

0.88

21.63

0.58

Table II Alachlor Plate Kit Calibrator Precision for 5 Runs

Cone. (ppb)

6/20/94 %B

6/21/94 %B

6/28/94 %B

7/6/94 %B

7/8/94 %B

Av. %B

% C V of

0.10 0.25 1.00 2.50

77.88 62.47 37.37 21.63

80.49 66.13 39.07 22.69

78.96 63.75 38.47 22.38

78.36 65.28 38.48 21.15

78.37 64.50 36.88 22.27

79.13 62.85 40.08 21.95

1.91 5.46 9.32 2.55

0

0

0

0

0

B

0

% B = (Absorbance of calibrator or sample/Absorbance of Negative control) X 100 %CW= (Standard Deviation/Mean) X 100 0


0

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Table III

Alachlor Plate Kit


Summary of Accuracy and Precision Data

Samples (ppb)

Average Cone. (ppb)

Cone, % C V

% Recovery

Average % Recovery

0.1 1.0

0.111 1.068

7.72 7.38

93-123 96-120

111 107

% B = (Absorbance of calibrator or sample/Absorbance of Negative control) X 100 % C V = (Standard Deviation/Mean) X 100 n=5 triplicate samples per level 0

Finally, in order to measure the precision and accuracy of the assay in drinking water samples, 11 water samples known to be negative for alachlor by G C analysis were spiked with 1.0 ppb alachlor and tested in the automated plate assay. Determinations were made in triplicate, and as summarized in Table IV, the percent recoveries range from 102.4 to 118.9. When the calculated concentrations for each of the triplicates were compared, the agreement was excellent, with CVs falling within the range of 1.5 to 6.9. This demonstrates excellent precision and accuracy for the determination of alachlor in drinking water samples.

Performance of the Assay for triazines In order to meet regulatory requirements in Europe, a highly sensitive triazine assay was developed to achieve detection levels below 0.1 ppb. European regulations set a limit of 0.1 ppb for any one compound, therefore the highly sensitive triazines assay was designed to detect atrazine levels ranging from 0.01 to 0.5 ppb. Table V shows the calibration curve using this triazines assay run on the automated system. The calculated % B 0


17. YOUNG ET AL.


values were all within the recommended ranges of 76-90% for the 0.01 ppb calibrator, 51-66% for the 0.05 ppb calibrator, 38-54% for the 0.1 ppb calibrator, and 20-27% for the 0.5 ppb calibrator. This information is included in the kit insert, and is used as a quality control check for assay performance. The calibrators were run in duplicate and gave excellent precision, with the %CVs for absorbance values ranging from 0.1 to 3. The data in Table VI demonstrates the stability of performance specifications of the triazines high sensitivity plate kit when run on four separate occasions, covering a period of 2 weeks. The % B values remain quite constant, yielding %CVs from 1.16 to 5.52.


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Table IV

Precision and Accuracy of Alachlor Determinations in Fortified Environmental Water Samples

Drinking Water Sample

Rep-1 (ppb)

Rep-2 (ppb)

Rep-3 (ppb)

Mean (ppb)

%CV

Mean % Recovery

1 2 3 4 5 6 7 8 9 10 11

0.957 1.167 1.172 1.105 1.111 1.091 1.070 1.065 1.191 0.957 1.008

1.055 1.207 1.131 1.016 1.092 1.000 0.968 0.997 1.020 1.028 1.042

1.134 1.193 1.048 0.993 1.073 1.056 1.059 1.016 1.170 1.087 1.040

1.049 1.189 1.117 1.038 1.092 1.049 1.032 1.026 1.127 1.024 1.030

6.9 1.39 4.62 4.65 1.42 3.57 4.43 2.79 6.76 5.19 1.51

104.87 118.90 111.70 103.80 109.20 104.90 103.23 102.60 112.70 102.40 103.00

% B = (Absorbance of calibrator or sample/Absorbance of Negative control) X 100 % C V = (Standard Deviation/Mean) X 100 0


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Table V

Triazines High Sensitivity Plate Kit


Calibration Curve on Automated System

Calibrator Value (ppb)

Absorbance (450 nm)

0.00 0.00 0.01 0.01 0.05 0.05 0.10 0.10 0.50 0.50

1.683 1.611 1.366 1.364 1.000 1.013 0.854 0.860 0.364 0.367

%B

0

Mean % B

81.70 81.58 59.81 60.59 51.08 51.44 21.77 21.95

Absorbance %CV

0

100.0

3.09

81.64

0.10

60.20

0.91

51.26

0.50

21.86

0.58

Table V I

Triazines High Sensitivity Plate Kit Calibrator Precision for 4 Runs

Cal. Cone. (ppb)

4/26/94

0.01 0.05 0.10 0.50

82.66 61.44 51.07 24.38

4/26/94

5/10/94

5/11/94

Average %B

% C V of Absorb.

82.26 59.90 49.34 23.42

1.16 3.31 3.02 5.52

0

83.54 61.47 49.93 24.98

81.86 60.09 49.37 22.43

80.97 56.60 46.99 21.89



17. YOUNG ET AL.

189


In order to further challenge the automated assay, and rriimic a real-life situation where actual water samples would be placed randomly throughout the plate, an experiment was designed to run 11 replicates of samples placed randomly over two entire microtiter plates. Assay standards were always placed in the first two columns of the plate, tap water or Milli-Q water samples spiked with 0, 10, 20, 100 or 500 ppt atrazine were randomly distributed throughout the remaining wells in the two full plates. The two plates were run simultaneously in the automated system. Accuracy and precision results are shown in Table VII. The % C V values for the concentrations are somewhat higher than seen in previous experiments, especially at the 10 ppt levels. Since % C V is calculated by dividing the standard deviation by the concentration, very low concentrations tend to make the number larger. If a % C V value of 28.2 is considered unacceptably high, then the detection limit of this assay could be set at 20 ppt rather than 10 ppt. This value is still 5 times lower than the European regulated level of 100 ppt. Table VII Triazines High Sensitivity Plate Kit Accuracy and Precision of Fortified Samples Placed Randomly in Two Microtiter Plates

Spiked Tap Water Samples Fortification (ppt)

% C V of Absorbance

% C V of Concentration

% Recovery Range

Average % Recovery

10 20 100 500

4.4 3.9 4.7 2.8

28.2 16.9 11.2 3.4

76-210 75-145 96-137 99-111

127 110 115 105

Spiked Milli-Q Water Samples 10 20 100 500

2.9 2.9 3.5 3.7

23.5 16.7 8.4 4.7

57-110 60-105 90-121 97-113

84 79 108 105


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Conclusion In summary, this study demonstrates that commercially available immunoassay kits used in conjunction with an automated plate reader can accurately determine atrazine and alachlor levels in spiked Milli-Q and tap water samples. The accuracy and precision are comparable to that generated by G C analysis (based on work generated for atrazine submission to the USEPA). The automated plate system used in this study accommodates two full microtiter plates, allowing for approximately 86 water samples to be screened at a time if calibrators and samples are run in duplicate. There is no sample preparation required for water analysis. Approximately 30 minutes of hands-on time is necessary for sample and reagent handling and loading into the instrument, and the run time for two plates is about 2.5 hours. This work demonstrates the use of ELISA technology and automated plate systems for the rapid, accurate, and cost effective screening large numbers of water samples for the presence of pesticides.

Literature Cited 1. Bushway, R.J., Perkins, L.B., Fukal, L . , Harrison, R.O., and Ferguson, B.S. Arch. Environ. Contam. Toxicol. 1991, 21, 365-370. 2. Bushway, R.J., Perkins, L.B., Savage, S.A., Lekousi, S.J., and Ferguson, B.S. Bull. Environ. Contam. Toxicol. 1988, 40, 647-654. 3. Bushway, R.J., Young, B.E.S., Paradis, L.R., and Perkins, L.B. Journal of AOAC Int. 1994, 77, 1243-1248. 4. Moody, J.Α., and Goolsby, D.A. Environ. Sci. Technol. 1993, 27, 2120-2126. 5. Goolsby, D.A. Thurman, E . M . , Clark, M.L., and Pomes, M.L. In Immunoassays for Trace Chemical Analysis; Editors, M . Vanderlaan, L . H . , Stanker, B.E. Watkins, and D.W. Roberts; ACS Symposium Series 451; 1991, pp 86-99. 6. Thurman, E.M, Goolsby, D.A., Meyer, M.T., and Kolpin, D.W. Environ. Sci. Technol. 1991, 25, 1794-1796. 7. Thurman, E . M . , Goolsby, D.A., Meyer, M.T., Mills, M.S., Pomes, R.L., and Kolpin, D.W. Environ. Sci. Technol. 1992, 26, 2440-2447. 8. Thurman, E . M . , Meyer, M . , Pomes, M . , Perry, C., and Schwab, A.,Anal.Chem. 1990, 62, 2043-2048.


Environmental Immunochemical Methods - American Chemical Society

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