U.S. EPA Contaminated Site Cleanup Information (CLU-IN)


U.S. Environmental Protection Agency
U.S. EPA Technology Innovation and Field Services Division

Immunoassay and Enzymatic Assays

Three categories of field analytical methods use biological systems to measure target analytes:

  • Immunoassays
  • Immunosensors
  • Enzyme-based assays that do not require the binding of an antibody to a target analyte as antigen

Immunoassay is the oldest, most well-known, and widely used of these three field analytical technologies. Although, in general, clinical chemistry has used immunoassay for many years, the approach began to be used in the environmental field in the early 1990s, when test kits became commercially available. Immunosensors employ the same basic biological technology as immunoassay, but the assay system is mounted on an optical fiber or membrane. As yet, immunosensors are not widely available, although systems have been developed for eventual field analytical use. While enzyme-based assays have been used in clinical chemistry for many decades, only now are they coming into use in environmental field applications, such as measuring toxicity and bioavailablity, which are not quantifiable by other field analytical technologies.

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Description

Immunoassay technologies use antibodies to identify and quantify organic compounds and a limited number of metallic analytes. The technology is used widely for environmental field analysis because the antibodies can be highly specific to the target compound or group of compounds, and immunoassay kits are relatively quick and simple to use. Antibodies have been developed to bind with a target compound or class of compounds. Sensitive colorimetric reactions, linked to the immobilization of the target compound by the antibody, are used to identify analyte concentrations. The determination of the target analyte's presence is made by comparing the color developed by a sample of unknown concentration with the color formed by the standard containing the analyte at a known concentration. The concentration of the analyte is determined by the intensity of color in the sample. The color intensity may be estimated roughly by the naked eye and compared to the color/concentration values on a chart, or it can be measured more accurately with a photometer or spectrophotometer and the measurement compared to a reference value.

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Typical Uses

Immunoassay is now a widely accepted field technology for the analysis of many organic contaminants and classes of contaminants (and at least one inorganic contaminant). Various immunoassay kits and methods are tailored to specific classes of environmental contaminants. For example, EPA has approved immunoassay methods for a number of contaminants, most of which are published in EPA SW-846:

EPA Published Immunoassay Methods

Method Number
Method Name
4010 A

Screening for PCP by Immunoassay

4015

Screening for Dichlorophenoxyacetic Acid by Immunoassay

4020

Screening for PCBs in Soil by Immunoassay

4025

Screening for Polychlorinated Dibenzodioxins and Polychlorinated Dibenzofurans (PCDDs/PCDFs)by Immunoassay

4030

Soil Screening for Petroleum Hydrocarbon by Immunoassay

4035

Soil Screening for Polynuclear Aromatic Hydrocarbons by Immunoassay

4040

Soil Screening for Toxaphene by Immunoassay

4041

Soil Screening for Chlordane by Immunoassay

4042

Soil Screening for DDT by Immunoassay

4050

TNT Explosives in Soil by Immunoassay

4051

Hexahydro-1,2,5-trinitro-1,3,5-triazine (RDX) in Soil by Immunoassay

4425

Screening Extracts of Environmental Samples for Planar Organic Compounds (PAHs, PCBs, PCDDs/PCDFs) by a Reporter Gene on a Human Cell Line

4500

Mercury in Soil by Immunoassay

4670

Triazine Herbicides as Atrazine in Water by Quantitative Immunoassay

Note: Methods 4025 and 4425 both require that samples be prepared using the traditional fixed laboratory, solvent extraction methodology typically employed to prepare samples for gas chromatography/mass spectroscopy (GC/MS) analysis. In addition, Method 4425 requires laboratory experience with cell cultures. However, time and cost savings may be realized by the use of these methods as an alternative to high resolution GC/MS analysis.

Complete versions of the SW 846 methods listed above can be found on-line at:
EPA SW-846 On-line

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Theory of Operation

Immunoassay takes advantage of the ability of antibodies to bind selectively to the specific physical structure of the target analyte present in a sample matrix, such as soil or water. Working much like a key and lock, the binding sites on an antibody attach precisely and noncovalently to their corresponding target analyte (also known as the antigen). Because binding is based on the antigen's physical shape rather than its chemical properties, antibodies do not respond to substances that have dissimilar structures. Compound-specific immunoassay kits have been developed to detect only the target analyte and its metabolites. However, class-specific kits have demonstrated the most use in the environmental field.

Click to see a figure that illustrates the interaction between an antibody and an antigen.

Enzyme-linked immunosorbent assay (ELISA) is used most in the field because of its speed, sensitivity, selectivity, long shelf life, and simplicity of use. Antibodies for the ELISA immunoassay have been developed specifically to bind to a selected environmental contaminant or contaminants. The selective response is used to confirm the presence of the contaminant(s) in samples. During the first step of the immunoassay, the walls of a test tube may be coated with the antibodies, or the antibodies may be introduced into the test tube on coated magnetic or latex particles. With either method of introduction, the quantity of antibodies and their binding sites is known. Second, some of the contaminant, or antigen, is combined with an enzyme that will react with a colorimetric agent to produce a color change that does not interfere with the antigen's ability to bind with the antibodies. The enzyme labels the antigen and allows for detection of the antigen's presence. The solution containing the labeled antigen is called the enzyme conjugate. When the colorimetric agent, or chromogen, is added to the solution, it reacts with the enzyme on the labeled antigen to cause a color to form.

During the analytical procedure, a known amount of sample and a known amount of enzyme conjugate are introduced into the test tube that contains the antibodies, and the target analyte present in the sample competes with the labeled antigen in the enzyme conjugate for a limited number of antibody binding sites. A chromogen then is added to the test tube to react with the enzymes on the labeled antigen to cause the formation of a color (click to see a schematic diagram of the process). According to the law of mass action, the more analyte present in the sample, the more enzyme conjugate the analyte will displace from the binding sites. The amount of bound conjugate is inversely proportional to the amount of analyte in the sample. The original concentration of the analyte can be determined by measuring the amount of enzyme conjugate bound to the antibody. Because the amount of bound enzyme conjugate determines the intensity of the color, the intensity of the color is inversely proportional to the amount of analyte present in the sample. The concentration of the target analyte can be determined by observing the color change visually or by using a photometer or spectrophotometer to measure the precise change in the color of the reaction. One immunoassay system for the measurement of mercury is not based on an inverse relationship between the concentration of the target analyte and the colorimetric response. This system shows a direct relationship between concentration and light absorbance.

The steps in the analytical process are described in greater detail in the section Mode of Operation, below.

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System Components

Most immunoassay kits include test tubes, the enzyme conjugate, the chromogen, other necessary solutions, and calibration standards. If the test tubes themselves are not coated with antibodies, a solution containing iron filings or latex particles coated with antibodies also will be included. Solid samples, such as soils and sediments, need to be prepared for analysis, and the materials necessary for these extractions are provided in kits that are purchased separately from the immunoassay kits. If some samples are likely to exceed the calibrated range of the analysis, sample dilution kits are also available from kit vendors. In addition to the basic supplies, some or all of the following accessory equipment may be needed for extraction and analysis, depending on the type of kits and techniques used:

  • Test tube rack or magnetic separation rack
  • Balance
  • Pipettes and tips
  • Timer
  • Differential photometer or spectrophotometer
  • Vortex mixer
  • Supplies necessary to dry very wet soil/sediment samples

The accessory equipment usually is not supplied with the collection and extraction kit or the test kit. Accessory equipment can be purchased or rented from the manufacturer. Most manufacturers will rent all necessary equipment as a package. Some of the items, such as a balance, pipettes, and pipette tips, can be purchased from another vendor. Fixed-volume, adjustable, and repeating pipettes often are needed. If the immunoassay test kits are to be used for a number of projects, it is more economical in the long run to purchase equipment than to rent it. The spectrophotometers usually can be operated on battery power.

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Mode of Operation

Although designed for field use, most immunoassay kits usually are used in a sample trailer, mobile laboratory, or other fixed location because of the amount of equipment required, the requirements for some kits to be stored under refrigeration, and the advantages of working in climate controlled conditions. The manufacturer provides step-by-step instructions for the analytical method to be used. Most immunoassay test kits follow a "cook book" procedure that is designed to allow a novice to use them proficiently. However, some training is required in the use of some test kits, particularly those intended for quantitative analysis. Training can be obtained from the manufacturer, often at the job site. However, a background in basic laboratory techniques, such as pipetting, and the generation of calibration curves and calculations is beneficial. The basic steps in the use of the kits are described in the two sections below.

Sample Preparation

Preparation may be required before samples can be analyzed with an immunoassay kit. Immunoassay techniques can be used to analyze liquid samples. For that reason, water samples may not require preparation before analysis. Soil samples cannot be analyzed directly and therefore must be prepared. Contaminants must be extracted from solid samples into a solution amenable to analysis. Preparation of each type of sample is discussed below.

While soil samples cannot be analyzed directly, water samples require no sample preparation before analysis unless they are turbid. When water samples contain sediment, they must be filtered through a 0.45-micrometer filter before they are analyzed. Permission from the regulatory agency to filter a sample is generally required.

When contaminants are in a solid media, such as soil, they must be extracted into a solution amendable to analysis. Typically, soil collection and extraction kits include the following: 1) soil collection devices, 2) filters, 3) an extract solution (often methanol), 4) vials for collecting the extract, and 5) diluent (buffer) solution. Soil collection and extraction kits are sold separately from the immunoassay test kit, and they differ slightly from one manufacturer to another. Collection and extraction kits may be packed in one or two small, easily portable cardboard boxes. A typical soil collection and extraction kit contains enough materials to collect and extract from 4 to 20 soil samples.

Five to 10 grams of a soil sample are weighed into a plastic soil collection device, and 10 to 20 milliliters of solvent, usually methanol, are added to extract the target analytes from the soil. The mixture then is shaken (or put on a vortex mixer) for one to two minutes and allowed to settle for a few minutes. Some manufacturers add steel balls to the collection devices to help break up the soil particles. After the mixture has settled, a filter cap is placed on the plastic collection device, and the extract is filtered into a vial. Then the extract is diluted with a buffer solution so that the matrix of the solution is similar to the standards used for calibration, the diluted extract is ready for analysis. Manufacturers provide step-by-step instructions with the kits to guide the user through the extraction process.

Very heavy, tight clay soils may not settle quickly and may take several filtration attempts to produce sufficient extract for analysis. In this instance, it is good practice to allow extra materials for sample extraction. Very wet soils or sediments may require extra preparation to remove excessive water before analysis. The manufacturers of the kits usually provide guidance on this issue. Gentle sample drying methods that compromise the analysis of non-volatile analytes include decanting standing water from the top of the sample and gently blotting the sample with paper towels or diapers.

Sample Analysis

If the antibodies are coated on the inside surface of the test tube, the sample and enzyme conjugate are combined directly in the test tube. If the antibodies are coated on magnetic particles or latex particles, a carefully measured amount of the solution that contains the coated particles is added to the test tube. Measured amounts of both the enzyme conjugate and the actual sample containing the target analyte are added to the test tube. The action is a timed incubation step. During the incubation, the analyte in the sample competes with the known amount of labeled antigen in the enzyme conjugate for the limited number of antibody binding sites. After incubation, the excess unbound enzyme conjugate is washed (removed) from the test tube.

The amount of the enzyme conjugate that remains in the test tube is measured through the use of a colorimetric reaction. An enzyme substrate and a chromogen are added to the test tube to cause the formation of the color. That action also is a timed step, after which a solution is added to stop the formation of color. Because the amount of bound enzyme conjugate determines the amount of color, the amount of color is inversely proportional to the amount of analyte present in the sample.

The color of the sample can be compared visually with a zero solution or blank for a "yes or no," or qualitative, result. A semi-quantitative result can be obtained by using either a color chart for visual comparison or a differential photometer to compare the degree of light absorbance of a sample with that of a standard or standards., A quantitative result can be obtained by generating a calibration curve of absorbance compared with a concentration obtained using a spectrophotometer, hand calculator, calibration standards, and a zero solution. The light absorbance of the sample can be read from the spectrophotometer and converted into a concentration using the calibration curve.

Each batch will include quality control samples such as a negative and positive control. Once the process has begun, all samples must be carried through the timed steps in equal fashion. That requirement limits the number of samples that should be analyzed simultaneously as it is very difficult to maintain the time schedule if a large number of samples are being analyzed.

Consistency is crucial to achieve the greatest possible precision. Pipetting reagents must be consistent for each sample, and the analyst must be careful to avoid cross-contamination. The procedure can be monitored for consistency and cross-contamination by duplicating standards, analyzing control samples, and analyzing method blanks. Novices will require practice to perfect their pipetting techniques.

Analysis Times

The time required for preparation and analysis of samples varies, depending on the immunoassay kit used, the sample matrix, the required detection limits, and the amount of precision and accuracy desired. Liquid samples, such as groundwater samples, can be analyzed directly or after one or several dilutions if the concentration of the analyte is above the kit's calibration range. Soil samples must be subjected to extraction to remove the target analytes into a solution. The total preparation time required could range from minutes to 2 hours or more per batch of 20 samples, and the time required for analysis typically ranges from 30 minutes to 2 hours.

Because of the wide variation among kits and preparation times, throughput of samples also can vary considerably. Throughput is lower for soil samples than for water samples because no extraction is necessary for water samples. The actual throughput depends on several factors: (1) the experience of the operator, (2) the size of the batches of samples analyzed together, (3) the exact brand of immunoassay test kit, (4) the number of dilutions required if a quantitative test kit is used, and (5) the number of quality control samples analyzed with the investigative samples. An efficient analyst could run as many as 50 to 60 water samples per day, while typical throughput of as many as 30 to 50 samples per day is common for soils because of the additional extraction step. If a number of complex dilutions are required, 20 to 25 samples in a day might be the maximum throughput. Other factors can affect throughput, as well. For example, if samples are being delivered to the analyst a few at a time, the analyst may have to wait until a complete batch of samples has been received before performing the analysis. All enzymatic reactions are sensitive to temperature, and cold conditions will slow the reactions and color development, reducing sample throughput.

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Target Analytes

Immunoassay kits are available for a wide variety of organic contaminants, including gasoline; diesel fuel; jet fuels; benzene, toluene, ethylbenzene, and xylenes (BTEX); polynuclear aromatic hydrocarbons (PAH); various individual pesticides and classes of pesticides; explosives and propellants; and individual Aroclors (polychlorinated biphenyls [PCB]) and mixtures of PCBs in soil and water. Currently, one immunoassay kit is available for an inorganic contaminant, mercury. Some kits are designed for classes of compounds (PAHs, for example), and will provide a concentration of total PAH, but will not indicate the concentrations of individual compounds. A test kit for carcinogenic PAHs also is available. Kits for various analytes are relatively slow to come to market because developing compound-specific antibodies is technically challenging and time-consuming.

Kits are available for a number of petroleum compounds and classes of compounds, including BTEX. Immunoassay test kits primarily measure lighter aromatic petroleum fractions, because straight-chain hydrocarbons do not elicit immune system responses. The test kits for petroleum hydrocarbons do not perform well in analyzing for heavy petroleum products with few aromatic components, such as motor oil or grease, or for highly degraded petroleum fuels, since the lighter aromatic constituents have been driven off.

Immunoassay test kits are available for numerous pesticides and herbicides, such as triazine herbicides; 2,4-dichlorophenoxyacetic acid (2,4-D); organophosphates; cyclodienes; carbamates; dichlorodiphenyl trichloroethane (DDT); and many more. Some test kits for pesticides respond to only one compound, while others respond to an entire class of compounds.

Immunoassay test kits can detect PCBs in soil, water, and wipe samples. Quantitative test kits have been developed for specific Aroclors, and several kits can measure the overall concentration of a mixture of Aroclors, i.e., total PCBs. Other kits can detect pentachlorophenol (PCP), commonly found in soil and water at wood treating sites. Immunoassay test kits that analyze for PCP also respond in various degrees to other chlorophenols.

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Performance Specs

Performance specifications include information about interferences, detection limits, calibration, quality control, precision, and accuracy.

Interferences

Several factors can interfere with the detection and quantification of elements in a sample. Some interferences, such as cross-reactivity, are inherent in the analytical method. Other interferences may be caused by outside factors, such as the sample matrix.

Cross-reactivity is the degree to which an antibody binds to a substance other than its target, which usually occurs when different compounds of similar structure can fit into an antibody's �lock.� The manufacturer provides information about potential cross-reactivity for compounds similar to the target analyte. The information is presented in terms of the concentration of another compound that produces a detectable response (or interference) when the immunoassay test kit is used. Sometimes, 100 to 1,000 times the concentration of another compound is necessary to cause an interference. However, in some instances, compounds other than the target analyte may give as great a response. The 4000 series of immunoassay methods described in SW 846 provide information on cross-reactivity.

It is particularly important to consider cross-reactivity when using immunoassay kits that analyze for classes of compounds. For example, a BTEX test kit will respond to all six BTEX components (including isomers) in different degrees but will not provide concentrations of individual compounds. However, the BTEX test kit is as sensitive to naphthalene as it is to the xylenes, and the xylenes produce the greatest response to immunoassay, followed by ethylbenzene, and then benzene. Cross-reactivity can be desirable. An antibody's ability to bind with similar compounds can make it possible to identify a number of similar constituents, such as carcinogenic PAHs, rather than individual compounds, thereby determining the overall amount of that class of contamination present at a site. Cross-reactivity is undesirable, however, when the user wishes to determine the concentration of a specific compound and avoid interference from similar compounds that may be present. Such interferences can cause false positive results. For example, if a user wishes to determine the concentration of benzene in soil or groundwater at a site contaminated with gasoline, immunoassay is not the best technology to choose for the analysis. This consideration can be particularly important when defining the extent of contamination or when performing a risk assessment. Thus, it is imperative to have some knowledge of the contaminants of concern at a site before an immunoassay test kit is selected.

Interferences can be introduced from the sample matrix. For example, when an immunoassay kit is used to test samples of contaminated clay soil, the results of the analysis may not be as reliable because the fine clay particles tend to adsorb contaminants to a greater extent than silty and sandy soils and are more difficult to break up for extraction. A good sampling and analysis plan that specifies rigorous sample extraction procedures and requires confirmatory sampling to assess whether the results of the on-site analysis are biased low helps manage such interferences and allow for their correction.

Many of the sample reagents, including the antibodies and chromogens, are highly sensitive to direct sunlight, which can break down the reagents or cause a change in the colorimetric reaction. For those reasons, most immunoassay kits cannot be used effectively in direct sunlight, and care must be taken to provide good shade when working outdoors.

Detection Limits

Detection limits for immunoassay often are comparable to or even lower than those for conventional analytical methods. Although the detection limits vary depending on the test kit manufacturer, target analytes, sample matrix, and interferences, kits are available that can achieve parts per million (ppm), parts per billion (ppb), and even parts per trillion (ppt) detection limits in water samples. Detection limits are higher for soils because extraction is necessary. In some cases, when the range of detection for a particular target analyte is actually too low to be useful, one or more dilutions may be performed. For example, if the action level for a contaminant is 50 ppm, it may be necessary to perform a 1:10 dilution of samples to be analyzed by a kit that has a detection limit of 50 ppb and an upper range of 5 ppm

Click on this link for a comparison of typical detection limits for common classes of compounds.

Calibration

Whether a quantitative or a semi-quantitative test kit is used, calibration standards are analyzed with each batch of samples. A standard contains a known concentration of the target analyte and is prepared for analysis in exactly the same way the environmental samples are prepared, ensuring that the standard is analyzed under the same conditions as the samples that are checked against the standard. For quantitative test kits, it is typical practice to generate a calibration curve, using three standard concentrations and a zero standard.

Quality Control

Ensuring that the data generated are of a known quality is vital to ensuring their usefulness. Quality control (QC) measures take several forms and can be performed in the field, during sample analysis, and after sample data have been collected. The amount and type of QC necessary will depend on the immunoassay test kit and the data quality objectives of the project. A much higher level of QC is necessary to produce definitive data. Typical QC measures, some or all of which may be used in immunoassay analysis for a given project or method, are discussed below and in the section in which precision and accuracy are discussed.

Whether a quantitative or semi-quantitative test kit is used, calibration standards are analyzed with each batch of samples to ensure that the standards are analyzed under the same conditions as the samples that are checked against the standards. For quantitative test kits, it is typical practice to generate a calibration curve, using three standards and a zero standard. The manufacturer will specify a minimum correlation coefficient, such as 0.99, that must be met. In the case of a quantitative test kit, the standards usually are analyzed in duplicate, and the manufacturer will specify the acceptable range of variation in absorbency or optical density.

Method blanks are samples taken during the various steps of the sample preparation and analysis process to monitor for (1) contaminants present in any of the disposable supplies or reagents; (2) cross contamination caused by poor pipetting; or (3) contamination caused by any other source, such as inadequate decontamination of reusable items. One method blank should be analyzed for every 20 samples. The method blank should not contain any target analytes in concentrations above the method detection limit.

Two analyses performed on the same sample are called duplicate analyses, and they are used to monitor the precision or reproducibility of the analytical technique. Duplicates should be analyzed at a frequency of one for every 20 samples. The variation between the results should be consistent with those provided by the manufacturer, or they must fall within a range determined by the analytical method.

Matrix spikes (MS) and matrix spike duplicates (MSD) are used to evaluate the extraction efficiency of the method and are another check of precision. The samples are prepared by spiking a known concentration of a target analyte into a sample representative of the matrix being analyzed. The spiking solution can be purchased from the manufacturer or from another reputable vendor.

Quality control measures such as MS and MSD are usually applied during fixed laboratory analyses and are not techniques routinely used during field analyses. However, these techniques may be employed in field laboratories to generate defensible data. As previously stated, the amount and type of QC necessary depends on the immunoassay test kit and the data quality objectives of the project. For example, data used to direct excavation would require significantly less QC than analyses verifying that remediation efforts have met established action levels.

Precision and Accuracy

Precision is a measure of the reproducibility of sample data from measurement to measurement, and it is affected by both the consistency of the test kit and the analyst's technique. Accuracy is a measure of how close the result of an analysis comes to the "true" concentration in a sample. There are several means of assessing an immunoassay sample's precision and accuracy.

Precision and accuracy are measures applied to quantitative immunoassay data. It is impossible to measure the precision or accuracy of semi-quantitative data reported as either greater or less than a given value, or within a range of pre-established values.

Precision is assessed by conducting several analyses of an environmental sample or a control sample and calculating the relative standard deviation of the sample results. That practice provides a measure of the variability of the results. The acceptance range for sample precision is determined by the data quality objectives for the project or is specified in the analytical method or the test kit vendor's instructions.

Control samples also are used to assess the accuracy of the immunoassay method and the kit being used. Control samples are solutions of known concentration, often supplied by the manufacturer. They are analyzed with each set of calibration standards before the samples are analyzed. The control sample will have an acceptance range that approximates the known concentration. If the method is to be considered accurate, the concentration obtained by the user for the control sample must fall into that range.

Performance evaluation (PE) samples, purchased from a specialist vendor, also can be used to check the accuracy of the method. PE samples are solutions of known concentrations of target analytes. While the user usually is aware that a particular sample is a PE sample, the user should not know the concentration of the analyte in it nor the acceptance range.

Confirmatory samples are collected from the same sample material that is analyzed on site, but they are sent to an off-site laboratory for formal analysis. The results of the on-site analysis are compared with the results of the off-site analysis to determine whether they are within the acceptable range. The acceptable range is determined by the analytical method, if applicable, or by the user. The purpose of a confirmatory sample is to judge the accuracy of the data obtained on site and allow for corrections, if necessary. To start with one confirmatory sample usually is submitted for every 10 to 20 samples analyzed on site. This number can be raised or lowered depending upon the results of the off site analyses.

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Advantages

There are numerous advantages to using immunoassay in the field, rather than formal analysis in a fixed laboratory. Speed, portability, relative ease of use, low cost per sample, real-time results, and the range of contaminants that can be analyzed are some advantages cited most commonly.

The detection limits for almost all analytes in water samples are lower than applicable maximum contaminant levels (MCL), and the detection limits for some analytes, such as pesticides, in water are an order of magnitude lower than MCLs. The detection limits in soil are comparable to, or lower than, those for conventional analytical techniques and lower than most action levels or remediation goals, as well.

All necessary supplies and reagents are provided in two or three small boxes that can be transported easily to a site in the trunk of a car or van. Many tests can be performed on a small table or a counter. No electricity is required, unless a photometer or spectrophotometer is used.

A beginner can learn how to use an immunoassay test kit in a day or less. Most people become proficient at using a test kit after analyzing just two or three batches of samples. The test kits are designed specifically for easy operation, although a background in environmental science and chemistry is helpful.

Depending upon the matrix, throughput as high as 30 to 60 samples a day is possible. Little, if any, sample preparation is required for water samples. The user therefore can generate data while field work is in progress, thereby reducing the likelihood that costly remobilization to a site will be necessary.

The typical cost of an analysis ranges from $10 to $30 per water sample and $20 to $40 per soil sample, plus the cost of labor. Because of the cost of labor and equipment rental, the cost per sample decreases as the number of samples increases.

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Limitations

Prior knowledge of analytes (contaminants present or suspected to be present) and potential interferences is necessary to select the correct immunoassay test kit and use it effectively. Obtaining that information may require the collection of samples for off-site analysis to determine the nature of contamination.

The petroleum hydrocarbon test kits do not perform well for heavy petroleum products, such as motor oil or grease, or for highly degraded petroleum fuels. Methanol is not the best extraction solvent for heavy hydrocarbons, and the immunoassay test kits primarily measure lighter aromatic constituents. In the cases of the analytes identified above, there is a potential for false negative results. As previously noted, there also is the potential for false positive results due to cross-reactivity.

When reagents require refrigeration, it is necessary to have a cooler or refrigerator on site.

It is preferable to have some degree of climate control when using immunoassay. Some reagents are sensitive to sunlight, so sometimes it is not practical to analyze samples outdoors, and wide fluctuations in ambient temperature can compromise the ability to use immunoassay kits in the field. All enzymatic reactions are temperature dependant, and proceed very slowly at temperatures below 50�F and rapidly at temperatures above 80�F. Data collected from an immunoassay system giving a sluggish response during the cold temperatures encountered on a cold spring morning may not be comparable to data collected later in the day when temperatures have risen considerably. Care should be taken to ensure that all test and quality control samples are analyzed at the same ambient temperature.

While analysis with some kits can be accomplished quickly, analysis with other kits can be time-consuming to perform.

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Cost Data

The relatively low cost per sample makes immunoassay an attractive field analytical technology for a variety of contaminants and projects. When calculating the cost per sample, the cost of the required quality control samples should also be factored in. In addition, the costs of equipment rental versus purchase should be considered. Accessory kits may cost from $2,000 to $6,000. Many manufacturers sell kits that vary widely in cost depending on the target contaminant(s). However, excluding labor costs, the cost of analysis usually ranges from $10 to $30 per sample for water and $20 to $40 per sample for soil. While it is best to contact manufacturers directly for cost information, manufacturers may offer discounts for bulk orders. Labor costs vary according to the labor rate of the analyst and the time necessary to complete a particular analysis. Thus, the total cost per sample decreases as the number of samples increases.

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Additional Resources

A User's Guide To Environmental Immunochemical Analysis

Comparison of gas chromatography/mass spectrometry and immunoassay techniques on concentrations of atrazine in storm runoff

EPA Region I Immunoassay Guidance

USGS Immunoassay use in Herbicides, Methods Development and Bibliography

Immunosensors

Immunosensors are biological detection systems (biosensors) that are coupled to a signal transducer. Like an immunoassay, an immunosensor uses an antibody to recognize an antigen (an environmental contaminant). The antibodies in the immunosensor may be mounted on a membrane that can be inserted into a portable analyzer or on a fiber optic probe. The antigen/antibody coupling generates a signal, such as a change in electrical potential, that is measured by an electrochemical transducer. Changes in fluorescence, reflectance, or absorbance can generate signals that an optical transducer can measure. While immunoassay kits are discarded after one use as the binding between the antibody and antigen is irreversible, immunosensor antibody/antigen binding can be reversible, thereby enabling multiple uses. Immunosensors also may be used as continuous monitoring devices.

In the late 1990s, the Naval Research Laboratory developed two immunosensor systems to detect the explosives RDX and TNT in environmental media. One system employed a flow cell technique, with a membrane-mounted fluorescent displacement immunoassay. The other had a competitive fluorescent system located on a fiber optic probe. More information is available on the flow cell immunosensor system in Review of Field Technologies for Long-Term Monitoring of Ordnance-Related Compounds in Groundwater (2005) ERDC/EL TR-05-14 http://www.clu-in.org/download/char/trel05-14.pdf

Enzymatic Assays

Enzymatic test kits and biosensor sticks are now commercially available to determine whether drinking water presents a toxic hazard due to contamination with carbamate or organophosphate pesticides. Enzymatic test kits and biosensor sticks use the same basic technology to detect these contaminants, namely the inhibition of the action of the enzyme acetyl cholinesterase (AChE) on a substrate, acetylthiocholine (ACE). One test kit system uses the hydrolysis of ACE by AChE to react with 5,5'-dithiobis-(2-nitrobenzoic acid) with a resulting yellow color. If the action of AChE is inhibited by organophosphates/carbamates then less color is produced. The reduction in color produced by the addition of a drinking water sample to the enzyme system can be compared to the color of a negative control. The color of the negative control and test samples can be read on a photometer, or a visual comparison can be made. Another system links the inhibition of the enzyme/substrate reaction to a change in pH, which is measured using a pH meter.

The enzymatic test kit (colorimetric endpoint) includes freeze-dried enzyme, substrate, and all other reagents necessary to run the assay. Disposable pipettes and sample tubes are also included in the kit. The photometer is not included in the kit. Incubation steps are required in this assay, but they can be performed at room temperature (70�F � 20�F). Although this kit must be stored in a refrigerator, all reagents should be at room temperature before analysis. Further information on enzymatic assays is available at: http://www.epa.gov/etv/vt-ams.html#etk

Enzyme-Based Tests for the Bioavailability of Heavy Metals

Enzyme-based tests can measure the bioavailability of heavy metals. The amount of a heavy metal available to a biological system is known as its bioavailability, a parameter that is similar to, but not always equivalent to the solubility of the metal in water. Bioavailability is a useful measurement in determining the toxicity of a metal in environmental matrices.

Genetically modified bacteria are used as whole-cell biosensors capable of detecting the bioavailable fraction in various environmental matrices, such as soil, sediments, water, and leachates. These modified bacteria contain a contaminant-sensing gene, linked to a reporter gene that is capable of producing a detectable signal. The presence of a heavy metal produces a metabolic change in the bacterial cells and activates the production of the enzyme luciferase, which causes the bacteria to emit light. If no heavy metal is present, no light is emitted.

Test kits are commercially available for determining the bioavailability of mercury and arsenic. The kits contain all the bacterial suspensions and other reagents necessary to conduct 30 tests, but the kits do not include the luminometer. The luminometer can be purchased separately from the kit vendor. The kit vendor describes a simple procedure for these measurements, with few steps:

  • Introduction of the sample suspension into a cuvette
  • Addition of the bacterial sensor suspension to the cuvette
  • Two hours incubation at 37°C
  • Addition of the substrate to the cuvette
  • Half hour incubation
  • Read luminosity

More information on heavy metal bioavailability is available at Interactions between metals, anaerobes and plants - bioremediation of arsenic and lead contaminated soils. (2002) R.Turpeinen

Rapid Toxicity Testing

Rapid toxicity testing kits have been developed that determine if drinking water poses a toxic threat. These kits use enzyme systems isolated from bacteria or the enzyme systems within whole small organisms, such as freshwater crustaceans, bacteria, or algae. The enzyme systems are linked to fluorescent markers that emit light if the system is functioning. Toxins inhibit enzyme function and consequently depress the production of light. Rapid toxicity assays respond to a range of stressors, including botulinum toxin, cyanide, ricin, thallium sulfate, and nerve agents. However, the enzyme system is reacting to a toxic insult and not to a specific compound or class of compounds. If a sample was determined to be contaminated, further analysis would be necessary to determine the nature of the contamination. Rapid toxicity assays are generally intended to evaluate drinking water toxicity, but some test kits can be used on soils and sediments. The U.S. EPA Environmental Technology Verification Program has issued verification reports and statements on 15 rapid toxicity testing systems and these are available at: http://www.epa.gov/etv/vt-ams.html#rtts

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Verification/Evaluation Reports

Verification of the performance of site characterization and field analytical technologies is conducted through a variety of programs. Evaluation and verification reports from EPA's Superfund Innovative Technologies Evaluation (SITE) Measuring and Monitoring Program, EPA's Environmental Technology Verification Program (ETV) program, and the Department of Defense's (DOD's) Environmental Security Technology Certification Program are provided below.

Superfund Innovative Technologies Evaluation (SITE) Measuring and Monitoring Program
The SITE Demonstration Program encourages the development and implementation of innovative treatment technologies for remediation of hazardous waste sites and for monitoring and measurement. In the SITE Demonstration Program, the technology is field-tested on hazardous waste materials. Engineering and cost data are gathered on the innovative technologies so that potential users can assess the technology's applicability to a particular site. Data collected during the field demonstration are used to assess the performance of the technology, the potential need for pre- and post-treatment processing of the waste, applicable types of wastes and waste matrices, potential operating problems, and approximate capital and operating costs.

EPA's Environmental Technology Verification (ETV) Program
EPA's Environmental Technology Verification (ETV) Program verifies the performance of innovative technologies. ETV was created to substantially accelerate the entrance of new environmental technologies into the domestic and international marketplaces. ETV verifies commercialized, private sector technologies. After the technology has been tested, the companies receive a verification report that they can use in marketing their products. The results of the testing also are available on the Internet.

DoD's Environmental Security Technology Certification Program
The goal of DoD's Environmental Security Technology Certification Program is to demonstrate and validate promising, innovative technologies that target the DoD's most urgent environmental needs. These technologies provide a return on investment through cost savings and improved efficiency. Technologies that have been certified through this program are listed below. Links are provided to the web sites that provide the Certified Environmental Technology Transfer Advisory and the Certification Notice for the technologies.

Cost and Performance Report: Integrated Field Screening for Rapid Sediment Characterization (CU 9717) 2004.

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