DEVELOPMENT OF A RAPID ON-SITE IMMUNOASSAY SYSTEM FOR THE ANALYSIS OF GASOLINE IN SOIL AND WATER S.M. Miller, D.R. Stocker, J.H. Rittenburg, C.Twamiey, B.S. Lal, and G.D. Grothaus Agri-Diagnostics Associates 2611 Branch Pike, Cinnaminson N.J. 08077 ABSTRACT A simple immunoassay system has been developed for the rapid on-site analysis of gasoline in water and soil. The system consists of a disposable assay device coated with antibodies specific to gasoline components, and a small handheld reflectometer for ‘quantitation of the assay results. Soil samples are extracted with methanol prior to analysis while water samples can be analyzed directly in the assay. ‘The competitive immunoassay is performed by sequentially adding sample, enzyme-labeled reagent, and enzyme substrate to the surface of the device from dropper bottles, As each solution is absorbed into the device, it passes through the surface zone of immobilized antibody. Sample analysis can be completed in 15 to 30 minutes giving a visually observable color endpoint. Each assay device contains a negative control reference zone that is used for comparison to the sample zone. The assay device couples to the handheld dual beam reflectometer which compares the color intensity of the sample zone to that of the reference zone. The results can be displayed as percent inhibition or as actual analyte concentration extrapolated from a pre- programmed curve. The development and performance characteristics of this system for the analysis of gasoline in water and soil is described. Introduction Gasoline contamination of soil and groundwater has become a topic of increasing concem since the enactment of new federal legislation in 1985 regulating Underground Storage Tanks (UST's). With an estimated 435,000 leaking UST's in the U.S., considerable efforts are being made toward their removal and subsequent clean-up of the contamination (1). This has put an increased burden on analytical laboratories to provide the timely information needed to make critical management decisions. The hazardous nature of the gasoline components and their potential for leaching into the groundwater, further enhance the need for rapid, and accurate information. ‘The current methods for measuring gasoline contamination include analysis for total BTEX (benzene, toluene, ethylbenzene, and xylenes) or TPH (total petroleum hydrocarbons) by EPA approved laboratory methods, which were not specifically developed for analysis of petroleum contamination (2). BTEX are common constituents of gasoline and are the principal chemicals of concern for the health risks. Except for field GC's, existing field methods do not measure BTEX directly but measure a larger fraction such as total volatile organics or TPH through use of instruments such as hydrocarbon vapor analyzers. These instruments can assist in site assessment and remediation activities by giving on-site information. However, results with these instruments have been shown to correlate poorly with laboratory results (3). 73‘The emerging use of immunoassay technology in agricultural and environmental, analyses provides a means of addressing many of the limitations of classical laboratory techniques(4). Immunoassays rely on highly specific, animal derived antibody proteins and relatively simple apparatus to detect and quantify a wide variety of target materials in a broad range of matrices. The high specificity of the antibodies. and high sensitivity of the immunoassay methods can significantly reduce sample preparation times. Simple assay procedures, high sample throughput , and relatively inexpensive instrumentation can make these methods considerably less expensive than conventional laboratory methods. Additionally, immunoassays can be adapted to rapid, on-site testing methods that generate timely information enabling better informed decisions to be made. This paper describes the development of immunoassay-based, field analytical systems for the detection and quantitation of the BTEX components of gasoline in water and soil. The sensitivity, reproducibility and specificity of these systems are also discussed. Immunoassay Technology Immunoassays are analytical techniques based on the specific and high affinity binding of a group of inducible animal-derived proteins called antibodies, with particular target molecules called antigens. Antibody formation by higher vertebrates is remarkable both in the specificity of the induced antibody to its target and in the variety of organic molecules and macromolecules that are able to induce a specific antibody response. The primary binding between the antibody and the target antigen forms the basis of the immunoassay, and a wide variety of immunoassay "formats" have been developed to allow either visual or instrumental measurement of this primary binding reaction. The tremendous variety of immunoassay formats and reagent configurations presently being used in medical, veterinary, food and agricultural immunodiagnostics all represent different ways of visualizing the primary antibody antigen reaction. ‘Over the past 25 years this methodology has been successfully applied to many of the analytical challenges of the medical health care industry for rapid and accurate measurement of analytes such as hormones, microorganisms, therapeutic drugs, drugs of abuse, and tumor markers. The past 10 years has seen a rapid expansion of immunoassay techniques into forensic, veterinary, food and agricultural analyses. The potential of rapidly measuring a very minute quantity of a specific analyte from within a complex sample matrix, often with little or no sample clean-up, is one of the attractive features that has led to the widespread application of immunoassay. Antibody-producing cells generally respond to macromolecules and compounds with a molecular weight greater than 10,000 Daltons when those materials are recognized as foreign by the immune system. In general, compounds of the molecular weight of most environmental contaminants will not independently elicit an immune response. If those compounds are covalently attached to a carrier macromolecule, however, the immune system will respond and produce antibodies to the small molecule portion of the complex (hapten) as well as to other regions of the carrier. ‘The specificity of an antibody to a small molecule can be influenced to a large degree by the design of the immunogen used to induce antibody formation. The immunogen is constructed by covalently coupling the target compound or a related analogue to a carrier protein. In general, antibody specificity is highest for the part of the molecule furthest from the carrier protein. ‘Thus in synthesizing the immunogen itis possible to orient the small molecule in ways that will favor antibody specificity to particular portions of the molecule. Most small molecule contaminants in the less than 1000 Dalton molecular weight range only have one antigenic determinant and thus must be analyzed using a competitive immunoassay format. The direct competitive immunoassay configuration used for the immunoassays is described in Figure 1. 74A variety of solid phase formats have been developed to simplify the handling and visualization of the reagents in an immunoassay, each representing various solutions to different end-user requirements. The most common variables to consider in format selection include the environment in which the assay will be performed, throughput requirements, and level of quantitation required. The immunoassay systems described in this paper have been formatted into a flow-through, single use device for simplicity and field-portability. The results are visualized by means of an enzyme-substrate reaction that produces a colored end-point, hence the name enzyme immunoassay (EIA). Quantitation of this end-point is achieved through the use of a handheld reflectometer. Assays performed in these flow-through formats can often be completed in less than 30 minutes and are ideally suited for field use. Materials and Methods Preparation of Protein-Hapten Conjugates Several immunogens were prepared using derivatives of toluene, ethylbenzene, and ©, m, and p-xylene conjugated to either bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or chicken albumin (CA) (Sigma Chem. Co., St. Louis, MO.). The antiserum chosen for use in the immunoassay was produced in a rabbit immunized with a xylene derivative conjugated to BSA using the periodate method of Nakane and Kawaoi (5). Several enzyme conjugates were prepared using derivatives of toluene, ethylbenzene, and o, m, and p-xylene conjugated to either horseradish peroxidase (HRP) or alkaline phosphatase (AP) (Boehringer Mannheim Biochemicals, Indianapolis, IN.). ‘The enzyme conjugate selected for use as the enzyme tracer in the immunoassay was produced by conjugating a p-xylene derivative to HRP via an active ester reaction, The bridging chemistry selected for the enzyme tracer was different from that of the immunogen to eliminate bridge recognition by the antibodies. Antibody Production Rabbits and sheep received 1 mg intradermal injections monthly (1:1 with Freund’s complete adjuvant) and were test bled monthly approximately 10 days following each booster injection. Antisera obtained from each animal were screened in an indirect ELISA. procedure on 96 well plates coated with the various chicken albumin-napten conjugates. Additionally, antisera collected over several months from selected animals were pooled and purified by protein-A affinity chromatography. This purified IgG fraction was evaluated in a direct competitive flow-through enzyme immunoassay (EIA) format (6) using several of the enzyme-hapten conjugates. The antibody and enzyme tracer chosen for the ELA were selected based on sensitivity with a total BTEX standard (CLP-BTEX, Accustandard, Inc., New Haven, Conn.) and cross-reactivity with gasoline components. Immunoassay Optimization ‘The EIA procedure described in this paper is a direct competitive immunoassay performed on the microporous surface of a porous plastic detector (7). This disposable analyte detector has two discrete reaction zones containing latex particles that have been coated with affinity purified antibody. Liquid reagents applied to the surface are drawn into the detector by capillary action causing the reagents to pass through the antibody reaction zones, thus increasing the antibody reaction kinetics. Figure 1 shows a schematic of the direct competitive EIA. ‘The prepared test sample is mixed with the hapten-HIRP tracer and applied to the sample zone on the surface of the detector, where analyte present in the sample competes with the tracer for antibody binding sites. An equal volume of a negative control reference and tracer are added to the reference zone on the detector. Following a 75rinse step, 3,3',5,5'-tetramethylbenzidine (TMB), a peroxidase substrate, is added to each zone which reacts with the bound enzyme tracer to produce a visible blue colored endpoint, As the level of gasoline in the sample increases, the color intensity in the sample zone decreases as compared to the reference zone. This color is stabilized with a fixing solution and is quantitated using a dual beam reflectometer (quantimeter) which measures the amount of reflected light from the two reaction zones and computes the difference. The quantimeter can display this color difference as percent inhibition or as the actual analyte concentration (in ppb/ppm total BTEX) extrapolated from a pre-programmed standard curve. ‘The amount of latex bound antibody immobilized onto the reaction zones was optimized to give maximum sensitivity by limiting the number of available binding sites. ‘The hapten-HRP tracer concentration was optimized to give optimal maximum color development in the reference zone with assay times of ~ 10 minutes. Sequential addition of sample and tracer showed lower sensitivity than the simultaneous addition of the sample/tracer mixture. Other optimized parameters include pH, ionic strength, polarity, sample volume, flow rate and sample : tracer ratio. Sample Preparation ‘Water Samples were collected into 16 x 100 mm polypropylene tubes with no headspace. Particulates were removed by filtration through a piston type filter and 1 ml was collected in the calibrated chamber of the piston filter. The enzyme tracer was mixed with the filtered sample to prepare the sample for the immunoassay in the quantitative range of 250 to 3150 ppb total BTEX (referred to as Water Low Range). The range of the assay ‘was expanded by diluting the Low Range sample 10 fold with dH and again adding tracer (referred to as Water High Range). Soil Samples were measured quantitatively using an open-ended syringe as a coring device. The gasoline components were extracted by mixing the soil with an equal weight of methanol followed by shaking for 1 minute. This procedure has been adapted from an API method for collecting soil samples for methanol extraction (8). Following a short settling time, the extract was filtered with the piston filter and then diluted 10 fold into the assay buffer. Tracer was added to prepare the sample for the immunoassay in the range of 3.5 to 80 ppm total BTEX (referred to as Soil Low Range). The range of the assay was expanded by diluting the Low Range sample 10 fold into the assay diluent and again adding tracer (referred to as Soil High Range). Results and Discussion Immunoassay Performance Sensitivity and Reproducibility ‘The standard dose-response curves for analysis of BTEX by immunoassay are shown for both the water and soil systems respectively (Figures 2 and 3) in which BTEX standards were prepared in either water or methanol. ‘The minimum detection limit of the assays, defined as the level of analyte required to give 20% inhibition in the assay, were calculated by determining the minimum concentration that could be differentiated from a negative sample with 95% confidence (two standard deviations from the mean). The linear quantitative range of the assay is defined as the level of analyte required to produce between 20 and 80% inhibition. The quantitative range for the water assay system was 250 to 3150 ppb and 3 to 65 ppm total BTEX for the Low and High ranges, respectively (Figure 2). ‘The quantitative range for the soil assay system was 3.5 to 80 ppm and 80 to 930 ppm total 76BTEX for the Low and High ranges, respectively (Figure 3). The relative standard deviations (% cv) are similar to those obtained for GC / GC-MS methods 8020 and 8240 for BTEX components at around 30% (9,10). Standard deviation bars are shown for points on each of the respective dose-response curves. Specificity Since gasoline consists of a complex mixture of closely related components, it was expected that these BTEX immunoassay systems would react to different levels of the various aromatic components. Reactivity profiles for the BTEX soil and water immunoassay systems were determined for each of these components (Table 1). From this data, it was apparent that p-xylene was the most reactive of the BTEX components tested. This is not surprising since the immunogen used for antibody production was a xylene derivative. The data also indicate reactivity with some semivolatile components of gasoline (trimethyl benzene and naphthalene). This reactivity profile needs to be considered when comparing immunoassay to GC results for total BTEX in weathered samples, where the immunoassay could tend to give higher results due to the higher proportion of semi-volatiles present following the loss of the volatile components. This tendency toward positive bias when analyzing gasoline contaminated samples by immunoassay provides an advantage in field screening applications where negative or uncontaminated samples are being screened out. The possibility of obtaining a false negative immunoassay result is significantly reduced due to the high bias. The different reactivity profiles observed in the water and soil assay systems are attributable to the fact that the two systems are performed in different sample matrices. The soil assay matrix consists of 10% methanol which lowers the polarity when compared to the water matrix. The different solubilities of the various fuel components in water samples and methanol extracted soil samples will also influence the availability and thus the sensitivity of the immunoassay for the different fuel types. Further studies are underway to determine the level of reactivity with other substituted benzenes and polynuclear aromatics (PNA'), as well as the aliphatic components of gasoline. ‘Comparison to Laboratory Methods In order to compare the BTEX immunoassay systems to the laboratory GC, asoline contaminated water and soil samples, as well as several spiked samples, were split for analysis by each method. Water Samples The immunoassay results obtained on 47 groundwater samples collected from service station monitoring wells compared very well to the BTEX values obtained on split samples using EPA method 602 (GC-PID) (Figure 4). Using a cutoff of 250 ppb total BTEX, there were 19 samples confirmed negative and 27 confirmed positive by both methods. There was only one alleged false negative sample by the immunoassay method, but reanalysis of this sample by GC showed no BTEX present. Soil Samples ‘The immunoassay results for 48 soil samples collected from remediation sites and gasoline stations compared well to the laboratory GC result for these same samples. Using a cutoff value of 3.5 ppm total BTEX, there were 26 samples confirmed negative and 20 samples confirmed positive by both methods. There was only one false positive immunoassay result which was near the cutoff value, and there was only one possible false 77negative sample by the immunoassay method, but reanalysis of this sample by GC showed no BTEX present. In order to directly compare the immunoassay to GC results for a methanol extract, the total BTEX and weathered gasoline standards were used to spike methanol samples which were subsequently split for analysis by both the immunoassay method and EPA ‘method 8020 (purge and trap GC-PID). The correlation of total BTEX values obtained by both methods (R? = 0.98) for the BTEX spiked samples was excellent (Figure 5). The results show very good agreement between the methods. The correlation of total BTEX values obtained by both methods (R2 = 0.92) for the weathered gasoline spiked samples was also very good (Figure 6), The immunoassay gave a bias of ~50% higher values than the GC method for the weathered gasoline spiked samples. This bias is probably be due to the reactivity of the semi-volatile components of the weathered gasoline in the immunoassay. Overall, the results obtained using the BTEX immunoassay systems for water and soil compared very well to standard analytical methods. Subsample variation and sample volatility may affect results, hampering the ability to obtain directly comparable results. ‘The low frequency of disagreement indicates that no significant sample matrix interferences were presented by the range of water and soil samples tested. Very good correlations were obtained, although the immunoassay results tended to have a high bias compared to the GC results. This bias reduces the possibility of obtaining false negative results, which is an advantage in field screening applications. Conclusions ‘This paper describes the feasibility of utilizing an immunoassay system for quantitating gasoline contamination in water and soil samples. A field-usable enzyme immunoassay was developed which reacts with the BTEX, as well as some of the semni- volatile components of gasoline. Simple sample preparation procedures were also adapted from existing methods to allow users to easily prepare and analyze soil and water samples. Users were able to prepare and analyze up to 5 samples in 30 minutes or less. Using a handheld reflectometer, quantitative results were obtained for total BTEX in water and soil samples. Due to the simplicity of the procedures and equipment used, these systems provide a versatile field screening capability. ‘The immunoassay systems have a detection range of 250 to 3150 ppb total BTEX in water and 3.5 to 80 ppm total BTEX in soil, and these ranges were expanded to 65 ppm for water and 930 ppm for soil through a simple dilution step during sample preparation. Very good reproducibility was obtained over the entire detection range. ‘The immunoassay reacts with each of the individual BTEX components but the best reactivity is with xylene. Reactivity was also observed with some of the semivolatile components gasoline, including the trimethylbenzenes and naphthalene. This broad reactivity profile enhances the overall sensitivity of the immunoassay system to weathered gasoline, which may reduce the possibility of obtaining false negative results for contaminated field samples. Direct correlations of immunoassay to standard analytical GC results for contaminated field samples were very good. The immunoassay results correlated well with EPA method 8020 results for 47 contaminated water and 48 contaminated soil samples. Frequency of false negative results by the immunoassay method was <2%, providing a reliable method for determining gasoline contamination. Excellent agreement was also observed between the immunoassay method and EPA method 8020 for spiked methanol samples. Additional studies are underway to determine the performance of the immunoassay systems in the field by correlating field results with those obtained by the laboratory methods. 78BTEX water and soil immunoassay systems provide a fast and convenient means of screening for gasoline contamination on-site to quickly obtain information about the levels of contamination present. This provides a cost effective way to obtain the timely information needed to make decisions on determining excavation boundaries, delineating contamination plumes, locating monitoring wells, and monitoring remediation processes. REFERENCES 1, Federal Register, Vol. $2, No. 74, p. 12664 (1987), 2. Potter, T. L. Analysis of petroleum contaminated water and soil: An overview, in Petroleum Contaminated Soils, Vol. 2, Calabrese, E. J. and Kostecki, P. T., eds. Lewis Publishers, Chelsea, MI, p. 97, 1989. 3. Denehan, S. A., Denehan, B. J., Elliot, W. G., Tucker, W. A. and Winslow, M. G. Relationship between chemical screening methodologies for petroleum contaminated soils: theory and practice, in Petroleum Contaminated Soils, Vol. 3, Kostecki, P. T. and Calabrese, E. J., eds. Lewis Publishers, Chelsea, MI., p. 93, 1990. 4. Vanderlaan, M., Watkins, B. and Stanker, L. Environmental monitoring by immunoassay, Environ, Sci. Technol., 22, (3), p. 247, 1988. 5. Nakane, P. K. and Kawaoi, A., Peroxidase-labeled antibody, a new method of conjugation, J. Histochem. Cytochem., 22, (12), p. 1084, 1974. 6. _ Rittenburg, J. H., Fitzpatrick, D. A., Stocker, D. R. and Grothaus, G. D. Development of simple and rapid immunoassay systems for analysis of pesticides, in Brighton Crop Protection Conf. Proc., Lavenham Press Limited, Lavenham, UXK., Publ, p. 281, 1991. 7. Rittenburg, J. H., Grothaus, G. D., Fitzpatrick, D. A, and Lankow, R. K. Rapid on-site immunoassay systems, in Immunoassays for Trace Chemical Analysis, Vanderlaan, M., Stanker, L. H. and Watkins, B. E., eds., ACS Symposium Series No. 451, American Chemical Society, Washington, D.C., p. 28, 1991. 8. Parr, J. L., Walters, G. and Hoffman, M., Sampling and analysis of soils for gasoline range organics, in Hydrocarbon Contaminated Soils and Groundwater, Vol. 1, Kostecki, P. T. and Calabrese, E. J., eds., Lewis Publishers, Chelsea, MI, p. 105, 1991 9. American Petroleum Institute, "Evaluation of Analytical Methods for Measuring Appendix IX Constituents in Groundwater", API Publ. 4499, p. 28, July 1989. 10. U.S. EPA, Test Methods for Evaluating Solid Waste; Physical / Chemical Methods, SW-846, Third Edition, Office of Solid Waste and Emergency Response, U.S. EPA, Washington, D.C., 1986. 79Table 1: Reactivity profile of the various gasoline components and fuel types in the BTEX immunoassay systems for water and soil. Water Assay System Soil Assay System Analyte IC 50(ppm)!_%Reactivity2 IC 50 (ppm)!_% Reactivity? Total BTEX 0.90 100.0 18.0 100.0 Benzene 14.00 6.4 250.0 72 Toluene 2.20 41.0 72.0 Ethylbenzene 2.50 36.0 72.0 p-xylene 0.62 145.0 285.7 o-xylere 8.00 113 113 m-xylene 5.20 173 195 1,2,4Trimethylbenzene 1.25 72.0 45.0 1,3;5-Trimethylbenzene 20.25 44 26 n-Propylbenzene 1.30 68.2 $14 Naphthalene 0.12 750.0 102.9 Decane >100.00 <1.0 <18 Tsooctane >100.00 100.00 <1.0 <18 Gasoline (Shell SU2000) 2.75 32.7 52.2 Gasoline (Exxon regular) 1.40 64.3, 66.7 Gasoline (Shell plus) 1.60 56.3 563 Gasoline (Shell regular) 130 60.0 56.3 Gasoline (Mobile regular) 1.60 56.3 43.9 Weathered Gasoline 2.25 40.0 55.4 Diesel Fuel 3.25 27 8.6 Jet Fuel 6.75 13.3 >1000.0 <18 Kerosene >100.00 <1.0 >1000.0 <18 2-methyl hexane3 >25.00 <3.6 >250.0 <12 Methylcyclohexane3 >25.00 <3.6 >250.0 <7.2 Anthracene3 >75.00 <1.2 >1000.0 <18 Acenaphthene3 >3.00 <30.0 >1000.0 3.00 <30.0 >250.0 <7.2 1, ICS0 is the concentration of free analyte required to give 50% inhibition in the immunoassay. 2. % Reactivity = ICSO Total BTEX/ICSO test compound x 100 3. Compounds have limited solubility. 80‘Negative Control Zone, Sample Test Zone (or Sample without BTEX) sample with BTEX) ABR @ F4Ea ‘Add Sample ‘And Conugate “Antibody on Solid Suppor me % Color and Read Percent Inhibition ‘Concenustion of STER KEY: == seid Suppor ER enryme Lied BE Hate Cmca wo Sp FD coors tut of Ezyme Subse w= teenies — EB) seni pn Figure 1. Diagram of the direct competitive flow through immunoassay format utilized for the BTEX immunoassay systems for soil and water. 810 i 0 100 10 Water Low Range Assay © Water High Range Assty 1 10 100 Total BTEX (ppm) Dose-response curves for the Water Low and High range immunoassay system. Curves were determined by analyzing BTEX spiked dH70 in the immunoassay systems. Each data point is the mean of 5 replicates and error bars represent one standard deviation. 1D Soil Low Range Assay © Soil High Range Assay 10 100 1000 10000 Total BTEX (ppm) ‘Dose-response curves for the Soil Low and High range immunoassay system. Curves were determined by analyzing BTEX spiked methanol in the immunoassay systems. Each data point is the mean of 5 replicates and error bars represent one standard deviation, 82100 o Immunoassay Analysis ‘Total BTEX (ppm) Figure 4. 500 400 300 200 Immunoassay Result (ppm total BTEX) Figure 5. 20 40 GC Analysis Total BTEX (ppm) Correlation of immunoassay and GC results for 47 gasoline contaminated water samples. Separate VOA vials were analyzed by the BTEX immunoassay system for ‘water and by EPA method 8020 (purge and trap GC-PID). Results are expressed as total BTEX for both methods. Each data point may represent more than one sample, 6 80 Slope = 0.99 RY = 0979 nae 100 200 300 400 GC Result (ppm total BTEX) Correlation of immunoassay to GC results for BTEX spiked methanol samples. Spiked samples were split for analysis by the BTEX immunoassay system for soil and EPA method 8020 (purge and trap GC-PID). Each data point may represent more than. ‘one sample, 500 600 83Slope = 1.57 R= 0921 6 (ppm total BTEX) Immunoassay Resul o 100 200 300 400 500 GC Result (ppm Total BTEX) Figure 6. Correlation of immunoassay to GC results for weathered gasoline spiked methanol samples. Spiked samples were spit for analysis by the BTEX immunoassay system for soil and EPA method 8020 (purge and trap GC-PID). Each data point may represent more than one sample, 84