Environmental Immunochemical Methods - American Chemical Society

Chapter 18

An Evaluation of a Pentachlorophenol Immunoassay Soil Test Kit

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Alan Humphrey Environmental Response Team, U.S. Environmental Protection Agency, 2890 Woodbridge Road, Edison, NJ 08837

Pentachlorophenol (PCP) has been used extensively as a preservative in the wood treating industry. Nearly 1,400 wood preserving sites exist in the United States, 56 appear on the National Priority List (NPL) and hundreds more may have been abandoned. This study was conducted to determine effective utilization of the EnSys, Inc. semiquantitative immunoassay test kits for on-site screening where PCP soil contamination is a concern. Analytical results from the kit and a Gas Chromatograph/Flame Ionization Detector (GC/FID) instrument were compared to ascertain if the kits were performing to manufacturer's claims. The GC/FID data were compared to GC/Mass Spectrometer (MS) data to assure its quality. Statistical analyses were performed both on the kit and GC/FID data to compare extraction efficiencies, develop possible explanatory models, determine dilution errors, and identify sources of variation inherent in the kit itself. In order to identify possible sources of error in the kit, the time component and the quantitation ranges developed by the manufacturer that are associated with its operating procedures were examined. Errors associated with kit operation and temperature were also considered. Statistical analyses indicated a statistically significant (p = 0.03) higher extraction efficiency by the laboratory method vs. the kit method; no statistically appropriate model could fit the GC/FID with the kit data; no statistically significant difference (p = 0.28) was observed between the GC/FID and the GC/MS data; and a good linear relationship was evident between the GC/FID and GC/MS data (r = 0.89). The EnSys, Inc. PCP semiquantitative immunoassay test kits appear to be an effective screening tool for PCP soil contamination determination when utilized properly. Recommendations are made to ensure more reliable data and improve the kit's performance and similar immunoassay field screening kits. 2

This chapter not subject to U.S. copyright Published 1996 American Chemical Society

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

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Pentachlorophenol (PCP) has been used for over 50 years in the preservation of wooden utility poles and pilings and contamination in soil is common on wood treating sites. A recent listing of the wood-treating industry indicated that nearly 1,400 wood-preserving sites exist in the United States, of which more than 700 are inactive. Fifty-six wood-preserving sites appear on the U.S. E P A Superfund National Priority List (NPL); hundreds more may also have been abandoned^. EnSys, Inc. developed a semi-quantitative immunoassay-based analytical screening method designed to detect PCP in soil. The ΡΕΝΤΑ RISç™ soil test system was developed as an efficient way to locate and map the extent of PCP contamination, screen samples in the field prior to laboratory testing, measure the effectiveness of remediation technologies, and ensure that cleanup levels meet state and federal regulations^/ The immunoassay technique utilized by this test kit is known as ELISA (enzymelinked immunosorbent assay). The following study was conducted by the U.S. Environmental Protection Agency (U.S. EPA) Environmental Response Team (ERT) and its prime contractor, Roy F. Weston, Inc. under the Response Engineering and Analytical Contract (RËAC). U . S. EPA/ERT/REAC have performed almost 800 tests with the EnSys ΡΕΝΤΑ RISc, test kit at four sites over a 14-month period. The EnSys ΡΕΝΤΑ RISc. test kits have been used during extent of contamination studies, removal activities, and treatability studies. The data set utilized in this study was collected during Comprehensive Environmental Response Compensation and Liability Act (CERCLA) removal activities at a former wood-treating site. The site was chosen due to the relatively uniform sandy matrix which comprise the unconsolidated deposits, the considerable size of the data set, and its wide range of contaminant concentrations.

The purpose of this study was: •

To determine the usefulness of the kit under actual field conditions.

•

To determine and make recommendations on the proper use of the kit to meet different site objectives.

•

To statistically determine the accuracy of the kit.

•

To determine if there is error inherent in the kit.

Theory EnSys test for PCP is based on an analytical method in which an antibody recognizes and binds to a specific chemical or antigen. An antibody is a protein which can be designed to attach to a small organic molecule such as PCP at very low concentrations (ppb range) with a high degree of specificity. A n antibody is analogous to a lock in the sense that there is a unique antigen shape, or key, that fits


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Evaluation of PCP Immunoassay Soil Test Kit

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into it. However, this shape can be repeated on different analytes causing crossreactivity. The test is a competitive assay in which the specially designed antibodies will bind both with PCP molecules in the unknown sample and with molecules of a PCP-enzyme conjugate. The conjugate reagent is an enzyme to which molecules of PCP have been chemically attached. PCP molecules in the sample compete with the PCP end of the conjugate reagent for a limited number of antibody binding sites. The greater the number of sample-derived PCP molecules relative to enzyme-attached PCP molecules, the larger the proportion of antibody binding sites that are occupied by PCP molecules originatingfromthe sample. After an incubation period during which the competitive binding occurs, unbound PCP and PCP- enzyme conjugate molecules are washed away and color-change reagents are added. The enzyme part of the bound conjugate molecules catalyzes the oxidation of a colorless substance to a colored (blue) one. The reaction is stopped by addition of dilute sulfuric acid (blue solution turns yellow), and the results are interpreted in the EnSys photometer. Figure 1 illustrates this analytical process. The degree of color development at the end of the test is proportional to the number of PCP-enzyme conjugate molecules bound to the antibody sites. Since the developed color intensity is inversely proportional to the number of PCP molecules in the sample, the concentration of a chemical in an environmental sample can be easily determined^. METHODOLOGY Analytical ΡΕΝΤΑ RISc. TEST KIT The EnSys, Inc. ΡΕΝΤΑ RISç test kit that was studied contained the following: 4 sample extraction jars each containing 20 mL of methanol capable of extracting 10 grams of soil; 20 antibody coated tubes which are inserted into the photometer to measure the absorbance/transmittance of the solution; 12 buffer tubes containing a buffer solution to which a 100-μί aliquot of diluted extract is added; 8 standard tubes which contain known concentrations of PCP to be used as a reference absorbance when a differential photometer is used; 1 500 mL wash bottle containing a buffered wash solution; and 3 dropper bottles containing reagents identified as substrate "A", substrate "B", and "STOP," which is used to halt the reaction of the enzyme with the substrates. Depending on the site specific level of interest, standard tubes are provided at several concentrations by the manufacturer, usually from 5 to 500 parts per million. After dilution of the sample extract and the color reaction, a comparison is made with several different standard tubes, providing an approximate range of the sample PCP concentration. For example, the sample tube is compared with two standard tubes, 10 and 100 ppm, on the differential photometer. A negative reading is obtained by comparison with the 10 ppm standard and a positive reading for the 100 ppm standard, indicating the sample concentration is between 10 and lOOppm.


194


Conjugate

Tube 1

Tube 2

1. Components of ELISA Chemistry

|»φ*| Conjugate

ϋ Standard Conjugate I

aI Tube 1 Standard

Neg. Sample

|x *xx| Pos. Sample

ΙWΊ w

Conjugate I

Conjugate 1

1

Tube 2 Neg. Sample

Tube 3 Pos. Sample

2. Enzyme Addition Figure 1. EnSys ΡΕΝΤΑ RISc Analytical Process. (Reproduced with permission from ref. 4 Copyright 1991 Advanstar Communications, Inc.)


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. χ .

Tube 1 Standard

Tube 2 Neg. Sample

Tube 3 Pos. Sample

3. Incubation and Competitive Binding Reaction

y Tubel Standard 4.

Tube 2 Neg. Sample

Tube 3 Pos. Sample

Wash

Figure 1. Continued

Continued on next page Van Emon et al.; Environmental Immunochemical Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


196

^

Substrate c;hromcgen (TME A 0 •

(

\ ate

1 Substr (H,0J

Substrate Β

•

* \ ^B)

k

^

Tube 1 Standard

Tube 2 Neg. Sample

Tube 3 Pos. Sample2

5. Color Development

6. Read Sample Figure 1. Continued


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Photometers Differential Photometer The differential photometer used in conjunction with the EnSys ΡΕΝΤΑ RISc Kit is a specific purpose photometer set at a 450 nanometer wavelength which gives an immediate direct comparison of the optical absorbance of two samples. During analyses with the EnSys ΡΕΝΤΑ RISc. Kits, standards are supplied. Once the color development test is complete, both "standard" tubes are placed in the photometer, and the one with the greater amount of absorbance is used as the comparison standard. Then, samples are placed into the photometer with the single standard and the differential absorbance is read. Spectrophotometer Following analysis in the differential photometer, the samples were read in a Hitachi V-2000 Double-Beam Ultraviolet/Visible (UV/Vis) spectrophotometer. This spectrophotometer is used to analyze the samples against a calibration curve of standards run through the kits and transferred to a cuvet measured for absorbance at 450 nm. According to the BeerLambert Law, the absorbance of a given sample is proportional to the concentration of the analyte for a given absorption pathlength at any given wavelength^. This analysis will yield concentration values for a given sample and a measurable error. GC/FID Method 8270/SW-846 (6) was modified for field use, substituting a Gas Chromotograph/Flame Ionization Detector (GC/FID) for the Gas ChromatographMass Spectrometer (GC/MS). An HP 5980 Series 2 Gas Chromatograph, equipped with a Flame Ionization Detector (FID) detector and controlled by a 33 96A integrator was used to analyze the samples The instrument conditions were: Column

Injection Temperature Detector Temperature Temperature Program Injection Volume

Restek RTx-5 (crossbonded SE-54), 30 meter χ 0.53mm ID, 0.50 μπι film thickness 300°C 300°C 70°C for 2 min., 8°C/min. to 285°C., hold for 15 min. 2μΙ,


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The G C system was calibrated using a 19 component creosote mixture and PCP at concentrations which ranged from 5 to 100 μg/mL (except for 2,4,6-tribromophenol which ranged from 25 to 500 μg/mL). The concentration of the detected compounds was calculated using the following equation 1:

DFxA^xVt

w

RF^xViXWxD where: DF RF^ \ C V Vj W D u

t

= = = = = = = =

Dilution Factor Response Factor (unitless) Area of Analyte Concentration of Analyte (mg/kg) Volume of Extract (mL) Volume of Extract Injected (μι) Weight of Sample (g) Decimal Percent Solids

Response Factor Calculation The response factor (RF) for each specific analyte is quantitated based on the area response from the continuing calibration check as follows in equation 2: A,

(2)

RF =

where: RF A, L

= Response Factor for a Specific Analyte = Area of the Analyte in the Calibration Mixture = Mass of the Analyte in the Calibration Mixture (ng)

An average of five values, R F ^ (at the five concentrations), was used.

GC/MS An HP 5995C GC/MS equipped with a 7673A autosampler and controlled by an HP1000 RTE-6/VM computer was used to analyze the samples (Modified Method 8270/SW-846) (6).


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The instrument conditions were: Restek RTx-5 (crossbonded SE-54), 30 meter χ 0.32mm ID, 0.50 μιη film thickness 290°C

Column

Injection Temperature Transfer Temperature Source Temperature Analyzer Temperature Temperature Program

290°C 240°C

Splitless Injection Injection Volume

240°C 30°C for 3 min., 15°C/min. to 70°C., hold for 0.2 min.;8°C/min. to 295°C., hold for 12 min. Split time = 60 sec. 1 μΐ.

The GC/MS system was calibrated using five Base, Neutral and Acid Extractable (BNA) standard mixtures at 20, 50, 80, 120, and 160 μg/mL. The calibration range was validated by evaluating the System Performance Check Compounds (SPCC) and the Calibration Check Compounds (CCC) as outlined in the Contract Laboratory Program (CLP) protocol. Before analysis each day, the system was tuned to decafluorotriphenylphosphine (DFTPP) and passed a continuing calibration check when analyzing a 50 μg/mL standard mixture in which the responses of the SPCC and C C C compounds were evaluated by comparison to the average response of the calibration curve. The concentration of the detected compounds were calculated using the following equation 3 : D F x A ^ x V ,

A^xRFxViXWxD where: DF RF A„ A^ Ι C V V W D ώ

u

t

A

= = = = = = = = = =

Dilution Factor Response Factor (unitless) Area of Analyte Area of Internal Standard Mass of Internal Standard (ng) Concentration of Analyte ^ g / K g ) Volume of Extract ( μ ί ) Volume of Extract Injected ( μ ί ) Weight of Sample (g) Decimal Percent Solids

Response Factor Calculation


(3)

200


The RF for each specific analyte is quantitated based on the area response from the continuing calibration check as follows in equation 4: Α,χΙ*

(4)

RF = AfcXlc where: RF A c

Ak I

c

Ik

= Response Factor for a Specific Analyte = Area of the Analyte in the Continuing Calibration Check = Area of the Internal Standard in the Continuing Calibration Check = Mass of the Analyte in the Continuing Calibration Check = Mass of the Internal Standard in the Continuing Calibration Check

Statistics Four statistical analyses were run to evaluate the immunoassay kit, the GC/FID, and the GC/MS data. These were logistic regression, linear regression, pairwise comparison t-test, and Wilcoxon rank sum test. For all statistical analyses, the significance level was set at 0.05. The significance level is the probability of incorrectly rejecting the null hypothesis in a given statistical test, it is set a priori to running a test to ensure correctness. To determine the statistical significance of the analyses performed, a p-value was generated with each statistic. The p-value is the lowest level at which the significance level can be rejected; in this case, any p-value less than 0.05 would show that the test is statistically significant. All statistical analyses were run on SAS software V6.06(7,8). Logistic Regression Logistic regression analysis fits a model between categorical response data and explanatory data. It differs from typical regression analysis in that instead of the model predicting a value for the response data based on the explanatory data, it gives an associated probability of falling into a given category based on the explanatory dataf / In this case, a model is fitted between the immunoassay kit data and the GC/FID data. The statistical parameter of interest in this test is the Score Test for Proportional Odds Assumption^, which tests if the logistic regression model is appropriate in explaining the data. 0


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Linear Regression Linear regression analysis fits a linear model between a response variable and an explanatory model. The statistical parameter of interest in the linear regression analysis is the F test, which establishes if the model is statistically significant. If the model is statistically significant according to this value, the r value is examined. The r value gives the proportional amount of variability that is explained by the model. It ranges from 0, which is no variability explained by the model, to 1, which is all of the variability explained by it(8). From these criteria it can be determined whether the linear regression model adequately fits the data. 2

2

Pairwise Comparison This hypothesis test determines if the mean difference between two sets of data is significantly different from 0. One data set is subtracted from the other to get a data set that is made up of the differences. If the test does not indicate rejection of the null hypothesis, it does not mean that the data sets are equal, but rather that they are not significantly different from each other (8). Wilcoxon Rank Sum Test The Wilcoxon Rank Sum Test is a nonparametric alternative to the t-test for comparing two groups of data. The only assumption required for the test is that each observation is independent. This test is run when either sample size is small or the assumption of normality cannot be madefSj. STUDY Field Data Comparison GC/FID vs. Kit Several hundred potentially contaminated soil samples have been analyzed with the EnSys ΡΕΝΤΑ RISç test kit at four different sites and compared to GC/FID results for the same samples. All soil samples used in this study were thoroughly sieved and homogenized prior to analysis. Poor correspondence between the kit data and the GC/FID data at these sites posed a need for this study. GC/FID vs. GC/MS Twenty samples analyzed with GC/FID were also analyzed by GC/MS to assure the quality of the GC/FID data. In order to determine the different sources of error, the following variables were examined:


202



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Extraction Sample Dilution Color Reaction Antibody Tube Variability Operator Differences To study each independently, the variables were studied either separately from the others or while the others were held constant.

Extraction Efficiency To statistically compare the two different extraction methods used for the determination of PCP three site soil samples of different concentrations were each separated into eight equal aliquots. Four aliquots were extracted by Ensys and four were extracted by the GC (GC/FID and GC/MS) procedure. A total of 24 extracts were analyzed by GC/MS using Modified Method 8270 (6). These samples were extracted using the following procedures. •

EnSys Extraction: Ten grams of soil were placed into the plastic extraction jar containing 20 mL of methanol/water solution and shaken vigorously for 1 minute so any clumps were adequately dispersed. The samples were allowed to settle for 15 minutes and then filtered with a supplied filtration device.

•

GC/FID and GC/MS Extraction: Ten grams of soil were mixed with 10 grams anhydrous sodium sulfate and 40 mL of 1:4 acetone:methylene chloride in a glass extraction jar. The jar was placed on an orbital shaker for 30 minutes at 300 rpm. The extraction was repeated two more times with 30 mL portions of solvent. The extracts were combined and brought to a volume of 100 ml.

Sample Dilution The kits that were used most often contained dilution vials labeled 0.5 ppm, 10 ppm, 100 ppm, and 500 ppm PCP in soil. These vials contained deionized water with a volume of 2 mL, 2 mL, 1 mL, 0.5 mL, respectively. When a 10 gram sample is extracted with 20 mL of methanol and 100 μΐ aliquots are transferred sequentially into each of these vials in series, the extracts are being diluted at ratios of 1:20, 1:20, 1:10, and 1:5 with an overall dilution of 1:20 (0.5 ppm), 1:400 (10 ppm), 1:4000 (100 ppm), and 1:20000 (500 ppm) as illustrated in Figure 2. These samples all dilute to a concentration of 12.5 ppb where the immunoassay test kit operates.

Color Reaction Error A 50 ppb laboratory standard of PCP in water was run with the kit eight times. This procedure eliminated any extraction and dilution errors associated with the kit since no extractions or dilutions were performed. Other errors, such as operator, life


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expectancy of the kit, and vial differences, were eliminated by using the same operator, using the same kit, and by using optically matched spectrophotometer cuvets instead of the plastic tubes provided in the kits. A100 aliquot of a freshly prepared PCP standard was added directly to the buffer tubes. Then, the entire contents of the buffer tube was poured into a antibody coated tube and allowed to react for 10 minutes. After reaction the contents of the dilution tube were poured off and the tube was rinsed four times with buffered wash solution and patted dry. Five drops of "STOP" solution were added and the color development was read and the absorbance was determined. The contents of the tubes had to be diluted with 0.5 mL of deionized water to raise the level of the liquid so the light beam was able to pass through. This procedure was repeated and the reaction time was extended to 20 minutes. The resulting data from 20 tests was then statistically compared. Antibody Tube Variability The kit antibody tubes became scratched when the kit was used. These scratches can account for error in the absorbance readings of the samples. If the standard vial is scratched and the sample vial is not or vice versa, erroneous readings may be obtained. In order to determine the variability in the supplied vials, used vials were filled with deionized water and compared to a clear vial at 450 nm. Absorbance readings were taken for each vial. R E S U L T S A N D DISCUSSION GC/FID vs. KIT Logistic regression analysis determined that no appropriate model (p-value

Environmental Immunochemical Methods - American Chemical Society

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