Immunochemical methods for environmental analysis - American

il for Environmental Analvsis LI

Jeanette M. Van Emon U.S. Environmental Protection Agency Environmental Monitoring Systems Laboratory 944 East Harmon Ave. Las Vegas, NV 89109

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Viorica Lopez Avila Midwest Research Institute California Operations 625-BClyde Ave. Mountain View, CA 94043

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Concern over rising costs and increasing sample loads has led to the development of immunochemical methods for measuring environmental contaminants. Immunoassays are immunochemical detection methods based on a reaction between a target analyte and a specific antibody. Quantitation can be performed by monitoring a color change or by measuring radioactivity or fluorescence. Many immunoassays have been developed for environmental com pounds that are difficult to detect by conventional methodologies. The recent rapid growth in immunochemical methods is attributed in part to the availability of polyclonal and monoclonal antibodies for a variety of compounds of environmental significance. For example, specific antibodies have been developed for many pesticides (1-20), industrial chemicals (21-28), microbial toxins (29, 30), organophosphorus chemical agents (31), protein products from microorganisms (32), and various bio-

0003-2700/92/0364-79N$02.50/0 0 1992 American Chemical Society

(21-28), explosives (24), and pollut ants leaking from underground gasoline storage tanks (21).Another drivi n g force i n t h e development of immunochemical methods is the need for rapid and simple tests that can be performed on site without requiring sample transfer to a n analytical laboratory. Field- portable immunoassays enable rapid determination of target compounds needed to effectively direct hazardous waste site remediation and cleanup. Many field methods can be used by personnel unfamiliar with analytical chemistry methodologies. In this REPORT we will provide a brief overview of the major applications of immunochemical methods and describe some of the activities of regulatory agencies. This will be followed by a discussion of the important elements of a n environmental

immunochemical method-antibodies and haptens-and the various immunoassays available for both lab oratory and field use. Advantages and disadvantages of immunoassays will be addressed, and examples of method performance will be given. Finally, we will address potential applications of immunoaffinity techniques and conclude with future research areas for immunochemical methods. Immunoassays are used for the direct determination of target compounds in environmental samples; immunoaffinity chromatography is used for specific separations of target compounds in complex matrices. The same antibodies used in an immunoassay can be used to develop immunoaffinity techniques. Specific antibodies immobilized onto a chromatography column have made it

possible to preconcentrate specific compounds from environmental Sam ples before their determination. In this case, t h e sample extract is passed through the immunoaffinity column containing a n immobilized antibody and the target analyte is retained by the specific antibody. By altering physical or chemical condi tions, one can remove the analyte from the immunoaffinity column and quantitate it by either immunoassay or instrumental analysis. Recently this technique was used to detect aflatoxins. Other areas i n which immunochemical methods have found unique applications are antibody- based bio sensors (36, 37) and personal exposure monitors (PEMs). A biosensor is a device that contains a biological component (e.g., enzymes, antibodies, or cell receptors) intimately connected to a transducer (e.g., electrochemical, optical, piezoelectric, or t h e r m i s t o r s o l i d - s t a t e devices). Whenever an interaction occurs between the target analyte and the biological component of the biosensor, some property of the transducer is altered and subsequently measured. Vo-Dinh and co-workers (37) have reported a regenerable immunochemical - based fiber-optic sensor for detection of benzo[alpyrenetetraol. This type of device uses a n antibody that is immobilized at the sensing terminus of the fiber-optic probe and allows remote, in situ measurements. PEMs based on immunoaffinity can monitor breathing- zone air for specific compounds. Similar immu noaffinity sampling devices can be deployed to collect pesticide spray drift and to sample industrial effluents. The feasibility of direct monitoring of pentachlorophenol (PCP) using a soluble antibody contained in a permeable membrane tube is being investigated (38). Immunoaffinity PEMs have several important attributes distinct

ANALYTICAL CHEMISTRY, VOL. 64,NO. 2,JANUARY 15,1992 79 A

REPORT from conventional devices. The use of specific antibodies enables these de vices to selectively monitor for a particular compound or class of compounds. The high binding constants for the target analytes indicate that PEM collection efficiencies are high. Furthermore, PEMs can be analyzed in the field at the end of a monitoring period and regenerated for reuse.

Tab1 hemic environmental arallysis Compoun

Activities of regulatory agencies Several regulatory agencies have been involved in the development of immunoassay methods. Of special interest is the ability to screen negative samples cost-effectively. Major costs are incurred for sample handling and shipment as well as for extraction and cleanup before analysis. Immunoassays have been used successfully to identify negative samples in the field, thereby reducing the number of samples sent for laboratory analysis. Similarly, expensive instrument time can be used more effectively because negative samples are eliminated and other samples can be designated for in-depth analysis, if necessary. However, for quality assurance, a certain percentage of negative samples must be analyzed by a confirmatory method. Methods development at the U S . Environmental Protection Agency (EPA) encompasses both field-portable and laboratory-based immunoas says. The primary goal is to develop simple assays that can be used easily by field crews at hazardous waste sites to monitor cleanup activities. Because information is generated in real time, a tight sampling grid can be executed rapidly to discern the extent of contamination. Timely reme diation activities can' then be initiated and process control optimized by on-site immunoassays. Field immunoassays can also enhance pollution control by providing timely data on industrial and hazardous waste site effluents.

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EPA has undertaken immunoassay development for several target analytes, including polychlorinated biphenyls (PCBs); benzene, toluene, and xylene mixtures; nitroaromatic compounds; parathion; carbaryl; and other pesticides. Assay development is initially undertaken with synthetic samples before making an evaluation based on real - world environmental samples. Standard operating proto-

ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15,1992

cols are written in formats such as those found in EPA solid waste (SW846) protocols. The U.S.Food and Drug Administration (FDA) focuses primarily on immunoassays for detection of pesticide residues in food and feeds. FDA anticipates that immunoassays will increase the sample capacity of their monitoring programs. By using immunoassays in a way similar to the

EPA program, FDA believes t h a t these assays may be used as initial screens to determine whether resi dues are present. FDA has developed immunoassays for monitoring natural toxins such as aflatoxins. However, the lack of appropriate methods for food matrices has hindered their use. FDA now is developing methods for compounds such as phenamaphos and carbendazime (39). The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) is supporting immunoassay development for pyrethroids, chlorinated pesticides, and other

-" Figure 1. A 96-well microtiter plate.

ilossary Antibody-a protein produced response to an antigen that binds I

Antigen-a large, chemically complex molecule or hapten-carrier conjugate that can induce an immune response, resulting in the formation of specific antibodies. Clone-a group of homogeneous cells that are the ProQenv of a sinale

unless it is covalently-bound to a carrier molecule. A hapten can react with the specific antibodies produced in respons carrier conjugate. Hybridoma-a hybrid of two different parental cells that contains genetic material from both cells. Monoclonal antibody-a homogeneous antibody population derived from one SD dv-tx ing cell. Polyclonal antisera-antibodies obtained from sera, often a heterogeneous population of antibodies varying in specificity and affinity, resulting from many antibody-nrodilfiing cells.

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compounds. Both field-portable and laboratory- based assays are included i n FSIS monitoring programs. As with other agencies, FSIS adapts existing assays to fulfill their monitoring needs. Kaufman and Clower (40) of the FDA discuss benefits and several concerns regarding commercial im munoassay test kits. Of particular concern is the lack of sample preparation methods for matrices other than water. Although immunoassays usually require much simpler sample preparations than those for either GC or GC/MS, t h i s requirement must be verified rather t h a n assumed. For large - scale monitoring studies conducted by regulatory agencies, the common 96 -well microtiter plate format is ideal (Figure 1).Although immunoassays are traditionally per formed in this manner, some commercial kits are based on test tubes or cards, and the advantage of large sample capacity is lost. Furthermore, these formats are not conducive to standard curve generation; thus, quantitation of target analytes is difficult. For field applications, t h e compromise could be to use 12-well microtiter strips with a portable spectrophotometer. These problems are legitimate and, because the use of commercial test kits is still rather new, further evaluation and research will be required to solve them. Although immunochemical methods have many environmental applications, they must be thoroughly evaluated and tested before analytical chemists will accept them. The current limited use of immunochemistry is attributable partly to the lack of fully evaluated methods based on real-world samples. Groups within EPA, the U.S. Department of Agriculture, and FDA recognize their common interest and concerns regarding immunochemical methods, and these agencies are trying to establish joint guidelines for evaluating test kits. It is anticipated that acceptance of the common guidelines will assist developers i n meeting t h e needs of the regulatory community. Adherence to these guidelines will also provide a measure of quality assurance. Antibodies for environmental

contaminants Immunochemical methods rely on antibodies typically produced in rabbits, sheep, or goats for polyclonal preparations or in mice or rats for monoclonal preparations (see glossary). Antibody molecules a r e com-

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posed of equal numbers of heavy and light polypeptide chains held together by disulfide bonds. Antibodies a r e usually depicted as Y-shaped proteins capable of binding to one analyte molecule at the tip of each arm (Figure 2). Environmental contaminants of low molecular mass (under 10,000 Da) must be coupled to carriers such as proteins to stimulate antibody production. Frequently, a chemical functional ity such as OH, COOH, NH,, or SH must be introduced onto the target analyte to form a hapten. The hapten is then covalently conjugated to a carrier to form an antigen for the production of antibodies. Often the coupling functionality is placed at the end of a n alkyl chain to offset the hapten from the carrier. This spatial removal of the small hapten from the large carrier provides a better target for the immune response. The design and synthesis of the appropriate hapten and subsequent hapten-protein conjugate a r e key steps in the development of immunochemical methods for environmental contaminants. Structurally, the hapten must be closely related to the analyte so that the analyte will bind to the resulting antibodies. Strategies for designing and coupling haptens to carriers have been reviewed (41). Antibodies a r e known for their specificity and for possible crossreactivity. The design of the hapten and the point of attachment to the carrier greatly influence the selectivity of the resulting antibody. Antibodies may be developed to detect only the parent compound, the parent compound and its degradation products, or a series of related compounds. Figure 2 depicts a n antibody selective for the herbicide paraquat. As much as 89% cross-reactivity is seen in this immunoassay with ethyl paraquat, whereas diquat exhibits only 20% cross-reactivity (13).None-

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theless, the assay is still quite useful for determining paraquat in environmental samples because ethyl paraquat is not a commercially available product. Antiserum for a particular hapten-protein conjugate is a mixture of antibodies produced by various clones of antibody-producing cells. These polyclonal antibodies are het erogeneous with respect to binding affinity and analyte recognition, yet

Figure 2. Simplified model of a paraquat-specific antibody shows the basic structure of four chains of amino acids held together by disulfide bonds. Antibody selectivity is based on the sequence of amino acids composing the two identicalbinding sites. The structures of two cross-reacting compounds, ethyl paraquat and diquat, are also illustrated.

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they may provide excellent reagents for detection techniques such as immunoassays. Many commercial im munoassay test kits are based on polyclonal antibodies. Through hybridoma technology, a homogeneous antibody population can be obtained by fusing a n antibody-producing spleen cell with a myeloma tumor cell (42, 43). The hybrid cells are then selected based on the secretion of the desired specific antibody and the ability to grow in cell culture. Although the selection process is tedious, it enables the separation of a particular clone from other cells producing unwanted antibodies. Assuming the clone can be successfully cultured, a particular monoclonal antibody can be obtained in virtually unlimited supply. Thus monoclonals enable greater s t a n dardization of reagents and may facilitate implementation of methods. However, for many environmental applications monoclonal antibodies are a n unnecessary expense because they may not provide improved selectivity or sensitivity.

Immunoassaysfor environmental analysis The development of a n immunochemical method is quite complex. The appropriate hapten must be designed, synthesized, and conjugated to carriers. Strict immunization regimes must be adhered to, and the antibody must be characterized and purified. Finally, the method must be formatted and evaluated. Figure 3 briefly summarizes the development process for a typical immunoassay using paraquat as a n example. Because paraquat does not induce a n immune response, it had to be conjugated to a carrier protein. However, before the conjugation s t e p could be performed, a spacer arm (e.g., (CH,),COOH)) had to be placed on the paraquat molecule to facilitate conjugation to the protein molecule. Proteins used for conjugation include keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin. Following antigen preparation, immunizations were initiated. Polyclonal antibodies were detected three months after the initial immunization. These antibodies were configured into a quantitative enzymelinked immunosorbent assay (ELISA) to detect paraquat in different matrices. I n a typical l a b o r a t o r y - b a s e d ELISA, a solid phase (beads or the wells of microtiter plates) is coated with a hapten-protein conjugate. A


Target analyte C H , + N x N + C H ,

Hapten design

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Figure 3. Steps in the development of a typical immunoassay, using paraquat as an example. solution of specific antibody that has been mixed with standards or samples is then added. During a n incubation period, the analyte in solution and the hapten-protein conjugate immobilized on the solid phase compete for binding to the specific antibody. A wash with buffer removes excess antibody remaining in the solution and any antibody-analyte complexes that have formed. An enzyme-labeled secondary antibody is next added and will bind with the remaining specific antibody captured on the solid phase. Alkaline phosphatase and horseradish peroxidase are commonly used as labels. An enzyme substrate is added, causing the development of a color. Absorbance or the rate of color development can be measured by a spectrophotometer designed to accommodate microtiter plates. Quantitation is based on competition for antibody-binding sites. In this assay format, color intensity is inversely proportional to the concentration of analyte in the sample. In-depth reviews of these procedures can be found elsewhere (44-46). Although quantitative immunoassays may require incubation periods of 1-2 h per step, the 96-well microtiter plates make it possible to analyze several samples at one time. The great advantage of immunochemical methods is that the r e -

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Cross-section of microtiter well Antibody Analyte Enzyme-labeled analyte Chromogenic substrate Colored product of enzyme-su bstrate reaction

Figure 4. ELSA format of a field-portable immunoassay. (a) Antibody is adsorbed onto a microtiter plate or test tube. (b) The sample and a known amount of enzyme-labeled analyte are added. (c) The analyte and the enzyme-labeled analyte compete for antibody-binding sites. (d) A chromogenic substrate is added for color production. (e) Color intensity, which is inversely related to the amount of analyte in the sample, can be estimated visually or measured with a spectrophotometer.

agents can be used in many formats. The earliest immunoassay consisted of an immobilized antibody to which a solution containing a n unknown concentration of the target analyte and a fixed concentration of a radiolabeled analogue of the target analyte were added (47).After a suitable incubation period, the unbound materials were washed away, the radioactivity of the bound antibody was measured, and the amount of target analyte was determined. Because immunoassays are physical assays, they obey the law of mass action. Thus the greater the radioactivity of the bound antibody, the smaller the amount of analyte that reacts with it. Radioimmunoassay (RIA) is less desirable for environmental analysis because it is not as field-portable, the half-life of commonly used '1 is short, and there are hazards associated with handling radioactive materials. The use of enzymes as labels

has evolved to circumvent the problems of radioactivity and to improve field -portability. As previously stated, one popular format for environmental applications is the ELISA. This assay format can be optimized for speed, sensitivity, or selectivity. Whereas laboratory methods are optimized for precision and sensitivity, field methods emphasize speed of analysis. Field-portable methods may employ 8-well microtiter strips or test tubes as the solid support. As shown in Figure 4a, the analyte-specific antibody is immobilized onto the microtiter support. A wash solution containing buffer removes any remaining unbound antibody. To perform a n analysis, standards and samples are diluted with buffer and placed into the microtiter wells. A constant, known amount of enzymelabeled analyte is then added (Figure 4b). Alkaline phosphatase and horse-

radish peroxidase are commonly used as labels. Competition between the analyte in the sample and the enzyme - labeled analyte occurs for the immobilized antibody (Figure 4c). After a short incubation period, all unbound materials a r e removed by washing the wells with a phosphate buffer, and a n enzyme substrate and a chromogen are added to produce a colored end product (Figure 4d). Quantitation of the target analyte is based on competition between the target analyte in the sample and the enzyme-labeled analyte that was added. The greater the amount of analyte in the sample, the lighter the color produced a t the end of the assay. Conversely, if samples contain only a small amount of the analyte, a dark color is produced because more enzyme-labeled analyte can bind to the immobilized antibody. Absorbance can be either measured in a portable spectrophotometer or estimated visually (Figure 4e). As w i t h immunoassays, many standard curves of absorbance versus logarithmic concentration are used for quantitation. An initial analysis of several logarithmic dilutions can be performed to obtain an estimate of the sample concentration. A limited range of dilutions are then performed to actually quantitate t h e target compound. A new automated immunoassay method based on flow injection analysis has been developed to detect pesticides in water samples (48).The flow injection immunoanalysis (FIIA) format consists of specific antibodies immobilized onto a membrane. Reagents a r e pumped i n a timecontrolled manner through the sensi tized membrane. The analyte and a peroxidase-labeled form of the analyte compete for binding to the immobilized antibody. The enzyme product of a fluorogenic substrate is measured downstream by a fluorescence


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REPORT detector. The membrane is automatically changed after each assay. FIIA has the potential to monitor water supplies, pesticide runoff, and industrial effluents for pollution control (48).

The environmental compounds most amenable to immunochemical methods include those that are hydrophilic, nonvolatile, and stable in water. Compounds such as sulfonylureas, benzoylphenylureas, carba mates, bipyridillium herbicides, and protein products from genetically engineered plants and microorganisms are ideal candidates for immunochemical methods. Compounds that are lipophilic, volatile, and somewhat unstable are still amenable to immunochemical methods, but t h e expense, skill, and time required for method development may not be justifiable. Immunoassay evaluation Although immunoassays are based on biologically derived reagents, they are not bioassays. To develop a n immunoassay, one must fulfill the same requirements established for other analytical techniques. Performance must be well documented and must address issues such as method detection limits, method sensitivity, appropriate positive and negative controls, and interferences. Because immunoassays require little or no cleanup, time and cost of an analysis are minimized. However, the method performance may be affected by interferences from the crude sample preparation. A well-characterized assay will address matrix effects by providing comparative data of standard curves produced in buffer and in the environmental matrix. The offset of the curves can then be established for quantitating samples. Controls are necessary to quantify matrix effects and antibody cross-reactivity. However, because of the large sample ca-

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0 0 0 9

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0 Solid-phase extract Surface water

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Drinking water

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Ground water

Method 604 extract

0 0

Surface water

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Ground water

Drinking water

Figure 5. Results comparing immunoassay and EPA GC Method 604 for the determination of PCP in surface water, drinking water, and ground water. (Adapted from Reference 35.)

pacity inherent in immunoassays, it is easy to include extensive controls in the analysis. Very few studies using real-world environmental samples have compared the data obtained by immunochemical methods with those obtained by conventional methods such as GC or GUMS. The results of one such EPA study (28, 35) are summarized below. The assay in the EPA study was a n ELISA for PCP, based on a monoclonal antibody and performed in a 96 -well microtiter plate. The linear dynamic range of the method was 30-400 ppb for environmental water samples. The results of the ELISA method correlated strongly to those of the standard EPA GC Method 604 (Figure 5). Based on various environmental water samples, the immunoassay generated a 9% false positive rate (115 determinations) and no false negatives (192 determinations). A field-portable kit version of the assay described above uses polyclonal antiserum and is performed in a n 8-well microtiter strip. Semiquantitative results in the 3-40-ppb range for PCP can be obtained in 20 min. The kit was evaluated at a n EPA Superfund site known to be contaminated with PCP (35). Environmental water samples were analyzed in parallel by the kit in the field and by GC/MS i n t h e laboratory. A Spearman r a n k correlation coefficient of 0.93 (16 determinations, 95% confidence interval) was calculated for samples with concentrations between 5 and 60 ppm (35).A 2% false


negative rate and a 5% false positive rate were observed. The majority of the false positive results were at concentrations ranging from 3 to 7 ppb, near the detection limit. The kit was determined to be suitable for monitoring the effluent of a bioremediation process for PCP degradation. An immunoassay for the rice herbicide molinate was evaluated using environmental rice field water by Harrison and co-workers (11). The assay was performed in a 96-well microtiter plate and had a detection limit of 20 ppb. No sample preparation other than buffering and dilution was necessary. Split samples were analyzed by both the immunoassay and an accepted GC procedure. The immunoassay results correlated well with the GC method results but had a slightly high bias, largely attributable to matrix effects caused by minimal sample preparation. An important point in the evaluation was the cost-effectiveness of the ELISA: The approximate cost per sample for GC analysis in the molinate study was $50 (for in-house work done by the researchers) and $130-$200 (for an outside contractor). This cost estimate was based on analyzing 10 samples per day with no replicate analyses. I n contrast, the ELISA costs were estimated at $5-$6 per sample (in-house work), based on 40 samples per day, three dilutions per sample, and four replicate wells per dilution. Another ELISA that demonstrated good correlation with conventional GC/MS methods was a commercially

available method used by Thurman and co-workers (49) for the determination of triazine herbicides (i.e., atrazine, simazine, and propazine) in water. This procedure uses a n antibody-coated tube with a n enzyme conjugate prepared by covalently binding atrazine to horseradish per oxidase. The assay had a detection range of 0.2-2 pg/L with a precision of 15-20%. No false negatives were observed, and the technique was estimated to cost $15 per sample.

lmmunoaffinity techniques for environmental analysis Immunoaffinity chromatography. This unique separation technique has been used to isolate specific antibodies using immobilized analytes. Recent emphasis has been on the immobilization of antibodies onto stationary supports for the separation of small molecules. The anti-

body-coated support is packed into a column, and the solution containing t h e specific a n a l y t e i s p a s s e d through. During this step, the immobilized antibody captures the analyte, separating it from other components in the solution. The captured analyte can then be removed from the adsorbent by dissociating the antibody-analyte complex w i t h a buffer solution. At least two applications have been reported in the literature. In one, the ecdysteroid- specific antisera are covalently linked to Sepharose 4B and used to purify ecdysteroids from insects (50). In the other, aflatoxin immunoaffinity columns are used to remove aflatoxins from foods and animal feeds (51). There a r e other literature citations on the use of immobilized haptens for antibody purification or immobilized antibod ies for peptide purification (52, 53).

Soil or sediment 10 9

3. No conceni necessi lmmunoaffinity support

Acidic SiO, SiO, (1st column)

Acidic alumina (2nd column)

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HRGC/HRMS or immunoassay

Figure 6. Proposed immunoaffinity procedure (a) compared witn tne conventional procedure (b) for extracting PCDDs and PCDFs from soil or sediment.

Figure 6 a presents a potential sample preparation procedure for polychlorinated dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). These compounds are normally extracted from soil or sediment with toluene using a Soxhlet extractor (Figure 6b). The extract is then subjected to a very tedious cleanup procedure in which three different chromatographic columns-acidtreated silica gel on top of untreated silica gel, acidtreated alumina, and Carbopack C mixed with Celite-are used. The details of the method are given in EPA Method 8290. Using this method, it takes two days to prepare a sample extract for GC/MS analysis. Furthermore, the costs for analyzing samples containing pptr levels of PCDDs and PCDFs range from $1000 to $2000 per sample, depending on sample complexity. Much of the cost is associated with sample preparation. This analysis would be simplified significantly if, after solvent or supercritical fluid extraction, the rest of the sample preparation were performed on a single immunoaffinity column (Figure 6a). Assuming that suitable antibodies are made available in large quantities, three steps a r e necessary to develop such a method. The first step is the selection of the support and the immobilization of the antibody. A variety of supports are available, such as soft gels (agarose), cellulose, resins, polymers (vinyl and acrylamide), bonded-phase silicas, glass beads, and coated glass beads. Methods to immobilize antibodies onto various stationary supports include use of cyanogen bromide and other cyano- based reagents that react with hydroxyl groups on the support and amino groups on the antibody. No single system seems to work best for all antibodies, and the immobilization of antibodies is still a


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trial-and-error procedure. Two critical parameters to study are the loading of the antibody on the support and the binding activity of the antibody. These criteria are important because they influence the sensitivity and the selectivity of the immunoaffinity technique. The second step is the development of a solvent system that is compatible with the antibody yet still hydrophobic enough to extract compounds such as PCDDs and PCDFs from matrices such as soil or oils and lipids. If water were the matrix of interest, the material could be sampled directly. However, this is rarely the case for these particular analytes. Based on the octanol-water partition coefficient of PCDDs and PCDFs, these compounds would probably have a moderate- to-low solubility in a solution of 25% isopropyl alcohol in water, a mixture that many antibodies will tolerate. Surfactants t h a t are compatible with the antibodies have been used to facilitate the release of bound PCDDs and PCDFs from soil or sediments (23). The third step is the development of a n eluent to release the bound PCDDs and PCDFs from the immunoaffinity column. Elution w i t h strong salts, pH adjustment, or elution with organic solvents is the most commonly used method. In the case of this target group of compounds, elution with an organic solvent such as methylene chloride would be ideal because the next step is analysis by high-resolution GC/high-resolution MS (HRGCIHRMS). Alternatively, they could be eluted with a solvent compatible with the immunoassay and analyzed by that method. Immunoaffinity PEMs. T h e principle of immunoaffinity is being applied to develop a PEM that uses a specific antibody to capture organic compounds from ambient air for the direct measurement of vapors. Applications might include workplace 86 A

monitoring, indoor air studies, and determining when it is safe for workers to reenter a previously contaminated site. These PEM systems are being considered for detection of PCP, parathion, dinitrotoluene, trinitrotoluene, and chlorpyrifos (54).Another PEM design uses microdialysis tubing with specific antibodies im mobilized inside (38).The membrane tubing acts as a n air-to-liquid interface, allowing vaporous analytes to diffuse into the aqueous medium where they become bound to immobilized antibodies. At the end of a sampling period, the tube fittings can be attached to a syringe, and the packing can be rinsed with a n eluent to release the analyte. PEMs can be coupled with rapid, field- portable immunoassay methods to obtain real-time residue and exposure information. Because of their selectivity and ease of analysis, these PEMs may enable better protection against exposure to environmental contaminants. Reentry times into pesticide - t r e a t e d fields could be based on actual residue levels. Other immunoaffinity monitoring devices could collect pesticide spray drift or monitor indoor air for a c u t e o r chronic exposures.

Implementation in environmental laboratories Immunochemical methods offer great potential for rapid screening and quantitative analysis of virtually all classes of compounds for which specific antibodies can be obtained. However, despite this potential, conventional methods of analysis (e.g., GC with element - selective detectors) still dominate the field of environmental analysis. Among the reasons such methods have not quite made their way into environmental laboratories is the lengthy method development time, which is typically six to eight months after development of appropriate a n tibodies. When the assay fails, it is often difficult to diagnose the cause of failure. Most important are the regulatory barriers. Although the method may work well, the regulatory agencies may reject it if it does not meet certain acceptance criteria or has not been tested with realworld samples. Guidelines for the acceptance of immunoassay test kit applications, setup of collaborative studies, data review, and adoption of immunoassay techniques have been proposed by EPA (55), the Association of Official Analytical Chemists (now AOAC International) (56, 57), and FSIS

ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992

(58). For example, EPA guidelines address criteria such as quality control, standard operating procedures, documentation of assay performance, availability and shelf life of immunologic reagents, and immunoassay characteristics that the developer should consider before the start of the evaluation. Raw data must be provided to determine the extent of fulfillment of the criteria. If the submitted assay is well characterized, a single- or multilaboratory evaluation may be undertaken. For very mature and well- characterized immunoas says, a short laboratory confirmation followed by a field demonstration may be conducted. EPA can then make recommendations regarding the application of the assay. AOAC International gives specific guidelines for conducting a collabora tive study and also has a task force to deal with issues such as use of proprietary reagents, the amount of detail necessary for method description, and t h e validation required before making a protocol change. FSIS requires, among other things, prior information about the cost of the tests and assurances of at least a one-year supply of reagents before it will consider implementing t h e method into a monitoring program. A serious deterrent to the implementation of immunochemical methods is the perceived problem of reagent availability. Although problems exist, several immunoassays, ranging from qualitative test kits to quantitative laboratory methods, are commercially available from a variety of vendors. Many of these companies are rather small, but large chemical companies also are conducting research. Some large companies use immunoassays strictly for in-house appli cations, and others are exploring the possibility of commercialization. The implementation of immunochemical methods will undoubtedly be influenced by the participation of established chemical firms. Future immunochemical techniques

Recombinant antibodies. Polyclonal antibodies can provide excellent reagents for immunoassays. However, they cannot easily be used for immunoaffinity applications because of their heterogeneous nature. Monoclonal antibodies can offer improvements because of their homogeneous binding characteristics. However, monoclonal antibody pro duction is labor intensive and may not be cost effective for low-volume applications.

Recently published reports (59)describe the expression of antibodybinding fragments in the bacterium Escherichia coli using recombinant technology. Current hybridoma technology enables hundreds of clones to be screened; however, recombinant antibody technology may enable the screening of millions. Furthermore, site-directed mutagenesis allows the manipulation of antibody genes to produce antibodies t h a t would not occur naturally. Although this a p proach has yet to be applied to antibodies for environmental contaminants, it appears promising and may ultimately replace monoclonal antibodies. Characteristics such as binding affinity, tolerance to physical parameters such as pH, and sensitivity to temperature or organic solvents could be engineered for a particular application. The development of synthetic antibodies and the increased capacity to screen them dramatically increases the possibility of finding a n antibody with the desired characteristics. The technology could also be used to produce antibodies for difficult targets. Catalytic antibodies. Antibodies have been demonstrated to catalyze certain chemical reactions. These catalytic antibodies can be used to increase the rate of elimination, cycloaddition and isomerization reactions, and aldol and Claisen condensations (60).The ability to introduce other catalytic groups on antibodies is being pursued. Catalytic antibodies eventually may be used to prep a r e environmental samples for analysis. Antibodies could catalyze a particular cleanup or derivitization step. Future uses may even include t h e detoxification of hazardous wastes. Multiresidue immunoassays. The cross-reactivity of antibodies can be used for a multiresidue analysis of s t r u c t u r a l l y r e l a t e d compounds. Statistical models can be constructed based on information from a library of antibodies exhibiting different sensitivities and specificities for a series of structurally related compounds. The model could give the concentration for each compound in the series and the confidence intervals on t h a t estimate, based on the presence of competing compounds. Metals. Immunoassays to detect organometallic compounds would be very useful for monitoring drinking water sources. Methods could be developed based on metal chelate and host-guest complexation research currently being conducted for thera-

peutic and pharmaceutic purposes. An ELISA for detecting mercuric ions in water has been reported (61), and a limited effort for the organometallics is under way. Summary Immunochemical methods, which are usually simple to perform, are powerful analytical techniques that can answer many questions concerning environmental contamination. I m munoaffinity PEMs enable timely assessments of exposure and can help to ensure that safe exposure levels are not exceeded. The application of immunoaffinity chromatography to sample preparation can minimize the use of organic solvents and subsequently reduce associated disposal costs. Furthermore, immunoassays are increasingly being recognized as one cost -effective alternative to chromatographic and spectroscopic proce dures for analyzing environmental contaminants, and they can be used in the field for rapid screening. Field portability and t h e rapid generation of data can help determine the source and extent of contamination, the fate and transport of contaminants, and the effectiveness of remediation activities. Laboratory assays enable increased sample throughput over traditional analytical procedures and can be used for large exposure or epidemiological studies. Immunochemical methods will not replace existing analytical techniques, but they can augment current monitoring and measurement capabilities. The full potential of such methods has yet to be realized. Although the research described herein has been funded wholly or in part by the U S . Environmental Protection Agency, it has not been subject to agency review and therefore does not necessarily reflect the views of the agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

References (1) Rittenburg, J. H.; Grothaus, G. D.; Fitzpatrick, D. A.; Lankow, R. K. In Immunoassays for Trace Chemical Analysis; ACS Symposium Series 451; Vanderlaan, M.; Stanker, L. H.; Watkins, B. E.; Roberts, D. W., Eds.; American Chemical Society: Washington, DC, 1990; Chapter 3, pp. 28-39. (2) Brady, J. F.; Fleeker, J. R.; Wilson, R. A.; Mumma, R. 0. I n Biochemical Monitoring for Pesticide Exposure; ACS Symposium Series 382; Wang, R.G.M.; F r a n k l i n , C. A.; Honeycutt, R. C.; Reinert, J . C., Eds.; American Chemical Society: Washington, DC, 1989; Chapter 21, pp. 262-84.

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(l‘ij’ Harrison, R. 0.; Braun, A. L.; Gee,

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(14) Newsome, W. H. Bull. Environ. Contam. Toxicol. 1988. 36.9-14. (15) Wittmann, C.lHock, B. J. Agric. Food Chem. 1991,39,1194-1200. (16) Bushway, R. J.; Perkins, B.; Savage, S. A.; Lekousi, S. J.; Ferguson, B-S. Bull. Environ. Contam. Toxicol. 1988., 40.. 647-54. (17) Giersch. T.: Hock. B. Food Amic. Immunol. 1990,2,’ 85-97. (18) Schlaeppi, J. M.; Fory, W.; Ramsteiner, K. /. Agric. Food Chem. 1989,37, 1532-38. (19) Goh, K. S.; Hernandez, J.; Powell, S. J.; Garretson, C.; Troiano, J.;Ray, M.; Greene, C. D. Bull. Environ. Contam. Toxicol. 1991,46, 30-36. (20) Newsome, W. H.; Collins, P. G. Food Agric. Immunol. 1990,2,75-84. (21) White, R. J . ; Van Emon, J. M. “Evaluations of Fieldable Immunoassay Formats”; EPA Internal Report 600/X-91/022; Environmental Protection Agency: Washington, DC, March 1991. (22) Albro, P. W.; Luster, M. I.; Chae, K.; Chaudhary, S. K.; Clark, G.; Lawson, L. D.; Corbett, J. T.; McKinney, J. D. Toxicol. Appl. Pharmacol. 1979,50, 13746. (23) Vanderlaan. M.: Stanker. L. H.: Watkins, B. E. Environ. Toxicoi. Chem: 1988,7,859-70. (24) Eck, D. L.; Kurth, M. J.; Macmillan, C. In Immunochemical Methods for Environmental Analysis; ACS Symposium Series 442: American Chemical Societv: Washington, DC, 1990; Chapter 9, p i . 79-94. (25) Lopez-Avila, V.; Carlson, R.; Van Emon, J. M. EPA Internal Report for EPA Las Vegas under Contract 68-033511, in preparation. (26) Luster, M. I.; Albro, P. W.; Clark, G.; Chae, K.; Chaudhary, S. K.; Lawson, L. D.; Corbett, J. T.; McKinney, J. D. Toxicol. Appl. Pharmacol. 1979,50, 14755. (27) Newsome, W. H.; Shields, B. J. Int. J. Environ. Anal. Chem. 1981,10,295-304. (28) Van Emon, J. M.; Gerlach, R. W. Bull. Environ. Contam. Toxicol., in press. (29) Ramakrishna, N.; Lacey, J.; Candlish, A.A.G.; Smith, J. E.; Goodbrand, I.A. J. Assoc. Ofi Anal. Chem. 1990, 73, 71-76. (30) Pestka, J. J. J. Assoc. OR Anal. Chem. 1988,71,1075-81. (31) Buenafe, A. C.; Rittenberg, M. B. Y

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REPORT Mol. Immunol. 1987,24, 401-07. (32) Wie, S. I.; Hammock, B. D.; Gill, S. S.; Grate, E.; Andrews, R. E., Jr.; Faust R. M.; Bulla, L. A.; Schaefer, C. H. J. Appl. Bacteriol. 1984,57, 447-54. (33) Haugen, A.; Becher, G.; Benestad, C.; Vahakangas, K.; Trivers, G. E.; Newman, M. J.; Harris, C. C. Cancer Res. 1985945,4178-83. (34) Foiles, P. G.; Chung, F-L.; Hecht, S. H. Cancer Res. 1987,47, 360-63. (35) “Evaluation of the Westinghouse Bioanalytic Systems PCP Immunoassay”; EPA Project Report No. EPA/6OO/ X-90/ 146; Environmental Protection Agency: Washington, DC, 1990. (36) Hall, E.A.H. Biosensors; Prentice Hall: Englewood Cliffs, NJ, 1991. (37) Alarie, J. P.; Bowyer, J. R.; Sepaniak, M. J.; Hoyt, A.M.; Vo-Dinh, T. Anal. Chim. Acta 1990,236, 237-44. (38) Drinkwine, A.; Spurlin, S.; Van Emon, J. M.; Lopez-Avila, V. Proceedings

of the Second International Symposium on Field Screening Methods for Hazardous Wastes and Toxic Chemicals; ICAIR, Life Systems, Inc.: Cleveland, OH, 1991; pp. 449-60. (39) Clower, M., Jr., FDA, personal communication, 1991. (40) Kaufman, B. M.; Clower, M., Jr. J. Assoc. Ofi Anal. Chem. 1991, 74, 239-47. (41) Harrison, R. 0.; Goodrow, M. H.; Gee, S. J.; Hammock, B. D. In Zmmun o a s a p for Trace Chemical Analpis; ACS Symposium Series 451; Vanderlaan, M.; Stanker, L. H.; Watkins, B. E.; Roberts, D. W., Eds.; American Chemical Society: Washington, DC, 1990; Chapter 2. (42) Kohler, G.; Milstein, C. Nature 1976,

256,52-53. (43) Winter, G.; Milstein, C. Nature 1981, 349, 293-99. (44)Hammock, B. D.; Mumma, R. 0. In Pesticide Analytical Methodology; ACS Symposium Series No. 136; Zweig, G., Ed.; American Chemical Society: Washington, DC, 1980; pp. 321-52. (45) Engvall, E.; Jonsson, K.; Perlmann, P. Biochim. Biophys. Acta 1971,251,42734. (46) Van Emon, J. M.; Seiber, J. N.; Hammock, B. D. In Analytical Methods for

Pesticides and Plant Growth Regulators; Sherma, J., Ed.; Academic Press: New York, 1989; Vol. 17, Chapter 7. (47) Yalow, R. S.; Berson, S. A. J. Clin. Invest. 1960, 39, 1157-75. (48) Kraemer, P.; Schmid, R. Biosens. Bioelectron. 1991, 6, 239-43. (49) Thurman, E. M.; Meyer, M.; Pomes, M.; Perry, C. A.; Schwab, A. P. Anal. Chem. 1990,62,2043-48. (50) Reum, L.; Haustein, D.; Koolman, J. Zeitsch rifr Natu rforschungen 1981, 36c, 790-97. (51) Sharman, M.; Gilbert, J. J. Chromatogr. 1991,543, 220-25. (52) Vaks, B.; Mory, Y.; Pederson, J. U.; Horovitz, 0. Biotechnol. Lett. 1984, 6, 621-26. (53) Janis, L. J.; Regnier, F. E. Anal. Chem. 1989,61, 1901-06. (54) “Development of Antibody-Based Personal Exposure (PEM) Devices,” draft final report prepared for EPA Las Vegas under Contract 68-03-3511, August 1991. ( 5 5 ) Van Emon, J. M. In Immunochemical Methods for Environmental Analysis, ACS

The National Institute of Standards and Technology has developed a series of SRM’s to serve as calibrants, test mixtures, and standardization materials for Quality Control of analytical instrumentation and methodology.

Symposium Series 442; Van Emon, J. M.; Mumma, R. O., Eds.; American Chemical Society: Washington, DC, 1990; pp. 58-64. (56) Mastrococco, D.; Brodsky, M. J. Assoc. OftrAnal. Chem. 1990, 73,331-32. (57) J. Assoc. OftrAnal. Chem. 1989, 72, 694-704. (58) Fed. Regist. 1989,54, 33920-923. (59) Huse, W. D.; Sastry, L.; Iverson, S. A.; Kang, A. S.; Alting-Mees, M.; Burton, D. R.; Benkovic, S. J.; Lerner, R. A. Science 1989,246, 1275-81. (60) Harris, T.J.R. Tibtech 1991, 9, 3941. (61) Wylie, D. E.; Carlson, L. D.; Carlson, R.; Wagner, F. W.; Schuster, S. M. Ahal. Biochem. 1991, 194, 381-87.

Jeanette M. Van Emon is technical manager for the immunochemistry Program at EPA. She received her B.S. degree in envi‘ronmental studies fiom California State University at Hayward and her Ph.D. in agricultural and environmental chemistry fiom the University of California at Davis. She received the joint EPA and ACS Science Achievement Award in Chemistry and the EPA Bronze Medal for her work in applying pesticide immunoassays to assess human exposure. She also was a co-organizer of the first ACS Sjmposium on Immunochemical Methods for Environmental Analysis. Her research interests include development of recombinant antibodies, fate and transport of pesticides, and toxicity of natural products.

MEASUREMENTS and STANDARDS are important to everyone who needs quality. NlST has over 1,000 Standard Reference Materials that can help you calibrate instruments and check on measurement accuracy. For more information phone or write for a free catalog. Telephone (301) 9 7 5 4 S R M (6776) FAX (301) 948-3730

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Viorica Lopez-Avila is director of the California operations branch of Midwest Research Institute. Since earning her Ph.D. fiom the Massachusetts Institute of Technology in 1979, she has been involved in developing analytical methods to determine environmental pollutants. Recently she has been involved in the study of supercritical fluid extraction (SFE) of organic compoundsfiomsoils, and she plans to develop field-screening methods that combine SFE with immunoassays.

Immunochemical methods for environmental analysis - American

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