United States Environmental Monitoring

Environmental Protection Systems Laboratory

Agency Las Vegas, NV 89193-3478

E AP Project Summary

Development and Evaluation of a

Quantitative Enzyme Linked

Immunosorbent Assay (ELISA)

for Polychlorinated Biphenyls

Research and Development EPA/600/SR-94/112 September 1994

A 96-well, microplate-based enzyme

linked immunosorbent assay (ELISA)

for the quantitative determination of

PCBs (as Aroclors) in soil has been

developed and evaluated. The method

detection limits are 8.95 and 10.5 μg/

Kg for Aroclors 1248 and 1242, respectively. The ELISA was characterized for

potential cross-reactivity with 37 structurally related chlorinated benzenes,

anisoles, and phenols which might potentially be present as co-pollutants in

environmental samples. Cross-reactivity was found to be negligible.

Samples were extracted using a

methanol shake extraction procedure

derived from various field screening

methods, including the EPA FASP (Field

Analytic Screening Program) gas chromatography (GC) method for PCBs. Extraction efficiency was found to be

greater than 90%, as determined by extraction of carbon-14 radiolabeled

tetrachloro-biphenyl.

Three sets of real-world samples, obtained from Superfund sites and the

EPA National Enforcement and Investigation Center (NEIC) were analyzed using the quantitative PCB plate ELISA.

In addition, all samples were analyzed

by outside confirmatory laboratories

using standard EPA GC methods. Subsets of the samples were also extracted

using supercritical fluid extraction and

methanolic Soxhlet extraction. The results for the methanol shake extracts

appeared to be statistically biased away

from the confirmatory results. The

ELISA results for the SFE and Soxhlet

extracts, however, overlapped the GC

results, within the error limits of the

respective methods. The latter results

demonstrated that the quantitative PCB

plate ELISA can function in a highly

precise and accurate manner as a detection and quantitation device when

coupled to an efficient extraction procedure.

Introduction

This report details the development and

evaluation of a 96-well microplate-based

enzyme linked immunosorbent assay

(ELISA) for the quantitative determination

of polychlorinated biphenyls (PCBs) in soil.

Procedures carried out during the developmental stage are described, along with

accompanying performance characteristic

data. In addition, the analysis of three

sets of real-world soil samples, representing a wide variety of matrix challenges, is

reported.

 After the initial detection of polychlorinated biphenyls (PCBs) in the environment in 1966, mounting evidence led the

U.S. Environmental Protection Agency

(EPA) to classify them as suspected human carcinogens, due in part to their low

rate of degradation, their tendency to

bioaccumulate, and their carcinogenic nature. In 1976, the U.S. Congress banned

PCB manufacture, processing, distribution

and use, except for a handful of specific

and limited uses. As a consequence, analytical method development resulted in the

codification of a number of standard methods for PCBs. One such method, Method

8080, described in the EPA Office of

Solid Waste and Emergency Response

Manual SW-846, is representative of the

majority of PCB analytical methods in that

the method relies on rigorous overnight

Jeffre C. Johnson and Jeanette M. Van Emon
2

Soxhlet extraction, followed by gas chromatography (GC) for quantitation of PCBs.

Several factors have generated increasing interest in immunochemically-based

analytical methods, such as ELISA, for

PCBs. In addition to the spiralling costs

associated with regulatory compliance,

current toxicological research has re-awakened the controversy surrounding the actual carcinogenicity and toxicity of PCBs.

In an effort to expand the array of tools

available for PCB research, immunochemically-based methods are being looked at

to provide data under appropriate circumstances.

 As a result of the Superfund Amendments and Reauthorization Act of 1986

(SARA), site monitoring and assessment

will represent a large and possibly rising

financial burden directly related to the collection, transport, and analysis of PCB

containing samples. ELISA based methods have been shown to offer the potential for data outputs which are complimentary and in some cases comparable to

established methods at a significantly lower

cost on a per analysis basis. ELISA methods also show a distinct advantage with

regard to timeliness of data generation.

Several ELISA-based methods for PCBs

have become commercially available over

the past several years. In general, these

immunoassays are formatted to be used

only for determining whether a given

sample contains PCBs at a concentration

above or below a set threshold value,

although one currently marketed assay is

intended for quantitative use. As such,

these immunoassay kits are designed for

rapid result generation, low cost, and ease

of use by relatively untrained personnel.

The benefits of such design criteria have

become quite evident to EPA, which, under the Superfund Innovative Technology

Evaluation (SITE) Program, officially mandated the use of low cost, rugged, fieldportable methods to ease the burden of

using expensive, time-consuming, GC or

GC/MS methods for the characterization

of contaminated areas.

While the benefits of using commercial

immunoassay kits are evident, the data

which they generate is complimentary but

not comparable with current GC-based

methods for PCBs. Consequently, there

exists a large gulf between these analytical methods.

 In the broadest of terms, the report

describes work on an immunoassay aimed

at bridging this gap between the GC based

instrumental methods and the commercial

immunoassay kits. Such an immunoassay

procedure will provide data which are

quantitative and comparable to the GCbased methods for many applications, such

as site characterization, mapping concentration isopliths, and monitoring remedial

activities, while providing high throughput

analytical procedures which are, to a great

degree, as inexpensive, rapid, and simple

as the kit immunoassays.

Conclusions

Assay Performance

The quantitative PCB plate ELISA was

characterized over the course of assay

development and subsequent analysis of

three sets of real-world samples obtained

from EPA SITE demonstrations and regulatory activities. Based on the Aroclors

which contaminated these real-world

samples the bulk of data are focused on

Aroclors 1242 and 1248. Initial characterization data show that this assay could be

used for Aroclors 1254 and 1260 equally

well with similar performance characteristics. The remainder of the discussion centers around Aroclors 1242 and 1248.

The assay described in the current study

is intended for the analysis of PCB contamination in solid matrices such as soil,

sediment, clays and paper pulp, and thus

the samples required extraction prior to

analysis. The methanol-based shake extraction procedure employed during the

present study was chosen for its simplicity, and was based on extraction procedures common to a number of field methods. Preliminary extraction studies, with a

wide variety of matrices, using a radiolabeled tetrachloro-biphenyl suggested that

the extraction procedure would optimally

provide an average extraction efficiency

of 92% with a relative standard deviation

(RSD) of variation of ±4%.

Extraction of commercially available

“PCBs in soil” standard reference materials (SRMs), followed by quantitation with

the plate ELISA provided an indirect measure of extraction efficiency. ELISA results for Aroclor 1248 SRMs suggest extraction efficiencies greater than 90%, while

for Aroclor 1242, employing 5 PCB levels,

efficiencies ranging from 53% to 91% were

observed. In all cases for the 1242 SRMs,

the reported value is within the EPA defined advisory range as specified by SW846 Method 8080/81.

The assay had detection limits of 1.31

ng/mL for Aroclor 1248 with a σ of 0.9 ng/

mL and a detection limit of 1.6 ng/mL for

Aroclor 1242 with a σ value of 0.61 ng/

mL. The detection limit in soil (based on a

5 gram sample) is 9.0 ng/g, σ = 6.0 ng/g

for Aroclor 1248 and 10.5 ng/g, σ = 4.1

ng/g for Aroclor 1242 after correcting for

the dilution factor imposed by adding soil

extracts to assay solution. The assay had

a quantitation range of about 8 ng/mL to

200 ng/mL in assay solution, corresponding to soil concentrations of about 50 ng/g

to 1330 ng/g, or 0.05 mg/Kg to 1.33 mg/

Kg. Samples extracts containing greater

than about 1.3 μg/mL PCBs require appropriate dilution to bring the PCB concentration into the working range of the

assay.

The assay provided the long-term reproducibility required for use as a quantitative tool. Based on repeated measures

of Aroclor 1248 soil SRMs over a 6 month

period, determinations were carried out

with RSD’s for all SRMs of less than 10%.

Repeated measures of Aroclor 1242 soil

SRMs over a 3 month period provided

similar performance. Dependant on PCB

level, RSD’s ranged from 30% to 5%.

The quantitative PCB plate ELISA was

found to be highly selective for PCBs; it

exhibited very little cross-reactivity with a

large number of compounds which might

potentially co-contaminate environmental

samples and interfere with accurate measurement of PCB concentrations. The assay exhibited selectivity for PCBs which

will allow the plate ELISA to be used in

the presence of a wide range of commonly occurring chlorinated anisoles, benzenes and phenol co-pollutants. The 37

compounds which were studied in the preliminary development stage cross-react no

more than about 3% relative to Aroclor

1248.

Validation of ELISA

Performance with Real-world

Samples

Validation of ELISA using comparative

results obtained by standard instrumental

methods is based on several important

assumptions. Statistically, this procedure

can become confounded by sampling errors, sample preparation differences, and

inter-lab variation, even before variability

is introduced by true inter-method differences. Comparison of the quantitative PCB

plate ELISA soil sample results with results generated using standard methods

such as SW-846 Method 8080/81 is made

difficult by the fact that performance data

for the standard methods is typically limited to either solution phase measurement

data or a limited number of soil matrices.

This lack of availability of extensive soil

analysis performance data for standard

methods points out the difficulty of comparing results across soil samples; each

soil matrix may present new challenges a

particular method cannot meet. Extraction

procedures which work well for sandy soils

may provide irregular performance characteristics when applied to oily clay

samples or sediments.
3

ELISA Analysis of Kansas City

Samples

ELISA analysis of Aroclor 1248 contaminated clay samples obtained as

sample splits from the Kansas City, MO

Indian Creek Superfund site provided data

which are interesting considering the above

discussion. Using a paired t-test as the

basis for decision, it was found that the

PCB levels as reported by the CLP laboratory were not equivalent to the PCB

levels as determined by the quantitative

PCB plate ELISA. The average relative

percent difference was found to be 46%.

One problem with such an approach is

the implicit assumption that both the ELISA

and CLP reported values represent the

true mean for each respective method.

The large error bars for the methods suggest that this is not likely to be the case.

One undesirable alternative would be running enough replicates of each sample to

ensure the validity of this assumption. This

degree of rigor is possible with immunoassay, however, given the time and expense of the CLP analyses, this is not

practical.

Another problem is the distinction (or

lack thereof) between preparative and determinative steps in the data generation.

The ELISA and CLP methods employed

very different extraction procedures. It is

conceivable that most of the measured

difference between methods is due to

sample preparation alone.

Four alternative hypotheses can explain

the data. The first is: the quantitative PCB

plate ELISA is not accurate but the CLP

method is. The second is: some interferant

or interferants in the samples themselves

causes assay performance degradation.

The third hypothesis is: the quantitative

PCB plate ELISA and CLP method are

both accurate, but extraction performance

varied greatly between methods. Finally,

the fourth hypothesis is: there is a large

quantity of some non-PCB cross-reacting

species present in the samples which elevates the apparent concentration of PCBs

as measured by the quantitative PCB plate

ELISA.

The first hypothesis can be ruled out.

Data generated for the commercial soil

SRMs as well as quantitation of spiked

solutions shows that the ELISA can accurately measure PCB concentrations. The

second hypothesis can be ruled out as

well. Data collected during parallelism studies with the Kansas City samples show

that there are no significant non-specific

matrix contributions for the ELISA results.

The third hypothesis cannot be readily

discounted. Based on earlier work (Spittler,

1986), it might be suspected that extraction procedures employing methanol would

work better than hexane/acetone extractions as called for in the CLP method.

Hexane/acetone extraction as specified in

the CLP method may be optimal for a

mixture containing all the chlorinated

analytes covered by the method, but not

for the specific, more focused use of PCB

extraction exclusively. Of course, the

simple approach taken in a methanolbased shake extraction used for the ELISA

might offset the gain realized from substitution of methanol. Results for the SFE

extracts of the Kansas City samples illustrate these extraction issues. Using the

identical ELISA procedure, it was found

that the SFE extracts gave results which

converged toward the CLP results. The

SFE-ELISA results were equivalent to the

CLP results by a paired t-test (t = 0.8729,

p = 0.39), whereas the ELISA results for

the same samples extracted by the methanol shake procedure were not (t = 2.118,

p = 0.046).

The fourth hypothesis cannot be ruled

out easily either. If there are cross-reacting compounds, they are not commonly

occuring chlorobenzenes, phenols, or

anisoles. One possibility is the presence

of PCB metabolites, such as polychlorinated biphenylols, which would not be detected by standard methods, but which

may nevertheless be measured by ELISA.

There are certain applications for which

data provided by the quantitative ELISA

could prove very useful. The reported data

for the Kansas City samples demonstrate

the use of ELISA as a bridge between GC

methods and semi-quantitative immunoassay test kits. The majority of the samples

had concentrations well below 5 mg/Kg.

At this level, relative percent differences

of 100% may correspond to as little as

0.033 mg/Kg (the detection limit of the

CLP method). For example, to easily obtain a quantitative PCB result of 0.1 mg/

Kg, ± 0.1 may have great value when the

option is either GC analysis or a semiquantitative “less than 5 mg/Kg” result obtained through use of a commercial, semiquantitative ELISA. The quantitative PCB

plate ELISA allows for the measurement

of PCB concentration in a way which provides more information than the semiquantitative ELISAs currently available

commercially, while using an assay procedure of similar complexity.

ELISA Analysis of Allied Paper/

Portage Creek/Kalamazoo River

Samples

ELISA analysis of Kalamazoo samples

obtained from the Allied Paper/Portage

Creek/Kalamazoo River Superfund site in

Michigan provides further amplification

upon the points discussed above. The data

can be thought of as consisting essentially of two subsets, the low level samples

(PCB concentrations below approximately

30 mg/Kg) and the high level samples

(PCB concentrations greater than 30 mg/

Kg). For the low level samples, the bulk of

the ELISA and SW-846 Method 8081 results overlap one another within the 95%

confidence intervals of the respective

methods. For the high level samples,

ELISA and Method 8081 results overlapped in a similar manner after more

vigorous methanolic Soxhlet extraction

prior to ELISA analysis. ELISA analysis of

extracts obtained using a 20 min. shake

in methanol resulted in measured values

of PCB up to a factor of 6.5 lower than

ELISA results for methanolic Soxhlet extracts. Again, parallelism studies and spike

recovery data demonstrated that the quantitative PCB plate ELISA, as applied to

the Allied Paper/Portage Creek/Kalamazoo

River samples, showed high accuracy and

no assay degradation due to matrix artifacts. Thus, the potential utility of the quantitative PCB plate ELISA as a determinative method for PCBs in sediment, soil

and paper waste was demonstrated by

the results.

The results for the high concentration

samples illustrate the need for differentiating the sample preparation from the determinative step itself. The ELISA results for

the simple “20 minute methanolic shake”

extracts and the ELISA results for the

methanolic Soxhlet extracts are very different. Clearly, only the extraction efficiency plays a significant role in altering

the ELISA results.

The fact that the ELISA results for the

methanolic Soxhlet extracts are convergent with the CLP data (the ELISA results

appear to be slightly lower than the CLP

results with a calculated mean RPD of -

17%) gives rise to the hypothesis that the

quantitative PCB plate ELISA, as the determinative step, provided comparable data

to the GC, for these environmental

samples, provided that appropriate extraction procedures were used.

ELISA Analysis of NEIC Samples

The results from ELISA analysis of

samples obtained from the EPA Enforcement and Investigation Center (NEIC) illustrate a number of points. Analysis of

several serial dilutions of the sample extracts demonstrated that the assay was

not subject to performance degradation

due to matrix artifacts. The ELISA results

were generally higher than the corresponding GC results, with an average RPD of

37%.
4

The results for this data set raise the

issue of calibration, an issue which is universal to any determinative method for

Aroclors. The samples were characterized

by NEIC as being mixtures of Aroclors

1242/1254/1260. The apparent bias high

on the part of the ELISA may be due

wholly to selection of the calibration mixture. In addition, the PCB levels reported

by NEIC most likely have a built in bias,

due to analyst judgement calls on assigning peak patterns to the various Aroclors.

Taken as a complete method, 20-min.

methanolic shake extraction followed by

ELISA determination of PCBs appears to

have bias away from standard GC based

methods, at least statistically speaking.

The quantative PCB plate ELISA data for

the three sets of real-world samples contain more information than results which

may be generated employing commercially

available semi-quantitative ELISA-based

methods. Thus, the quantitative PCB plate

ELISA fulfills the goal of providing an easily performed method bridging the performance gap between GC methods and

semi-quantitative ELISA.

The real-world data indicate that extraction procedures play a major role in the

ELISA results. Statistically speaking, in

these studies, it is improper to compare

the accuracy of the ELISA determinative

step with the GC determinative step, because the extraction procedures confound

matters.

The data generated during development,

evaluation, and application of the ELISA

strongly suggest that the quantitative PCB

ELISA can function in an accurate and

highly precise manner when considered

as a “detection and quantitation device”

separate from the sample preparation itself.

Recommendations

The analyses conducted in the course

of the current study strongly suggest that

the PCB ELISA has great potential for

use as a determinative step in PCB analysis, in particular, when coupled with an

appropriate sample preparation procedure.

To ascertain the performance of ELISA as

a “detector system,” it will be important to

remove the statistical ambiguity resulting

when the data being generated by two

detectors (GC/electron capture detector

and ELISA) are actually the result of measurements on two distinct soil extracts.

The two soil extracts are very likely different in their PCB concentrations, and thus,

even in the best case scenario of 100%

accuracy in the measurement step, the

results will of course differ.

To remove the contribution of errors in

soil extraction, it is suggested that further

experiments be carried out in which

samples are extracted and portions of the

extracts are quantitated by both GC and

ELISA. Any extract cleanup procedures

should be carried out before splitting the

extract. Alternatively, a study design could

be structured such that analyses could be

carried out on extracts which had been

cleaned up as well as extracts which had

not been subjected to additional clean-up

steps, thereby allowing for checks on the

possible effects of the cleanup procedure.

Further work could be carried out to

allow unambiguous comparison between

the PCB ELISA and GC/ECD as

quantitation techniques. One possible scenario would entail re-extraction of the Kansas City and/or Allied Paper samples, followed by analysis using ELISA and GC

(in-house and/or CLP laboratory) of splits

derived from these extracts.

The results of this study suggest that

ELISA has the capability of providing useful data for certain applications. The indirect inhibition format was used because,

generally, it is one of the more sensitive

formats which can be chosen from the

myriad of ELISA formats. In addition, this

format prevents exposure of the enzyme

to potential interferants present in the original sample. The assay can be configured

to allow even greater ease of use. For

example, the equilibration times may be

reduced allowing a one day assay without

a substantial change in performance.

The plate ELISA format can be easily

automated using any number of the readily

available robotic plate ELISA instruments.

This would permit screening of large numbers of samples, and in addition, it could

allow for the carrying out of extensive

parallelism studies on a routine basis. Extensive quality assurance of the ELISA

data output could thus be ensured.

A note of caution is raised with respect

to simple or abbreviated extraction procedures. Typically, most of the commercially

available semi-quantitative ELISAs for soil

screening rely upon “quick shake” extraction procedures, enabling extreme streamlining of the entire ELISA based procedure. The experiences noted in the current study reflect the possible dangers in

assuming that these extractions always

perform adequately.

The quantitative PCB plate ELISA can

be used to measure PCBs with high accuracy and precision when coupled with appropriate sample preparation procedures.

Further utilization should include coupling

the quantitative PCB plate ELISA with efficient and potentially fieldable extraction

methods, such as supercritical fluid extraction. Additionally, the quantitative PCB

plate ELISA could be coupled with rapid

SoxtecTM type extraction procedures, potentially in mobile laboratories, enabling

rapid, relatively non labor-intensive measurement of PCBs. This would be an analytical scheme of high utility, and acceptance, as use of Soxtec type extraction

procedures for PCBs already has precedence in such methods as EPA SW-846

Method 3541, Automated Soxhlet Extraction.

Interest in the application of the quantitative PCB plate ELISA has been generated within a number diverse groups, such

as the EPA Great Lakes National Program Office and the Fish and Wildlife Service, National Fisheries Contaminant Research Center. In order to facilitate the

application of the quantitative PCB plate

ELISA, the ELISA procedure, coupled to

a suitable extraction procedure should be

subject to peer verification through such

avenues as the Association of Official Analytical Chemists (AOAC) Peer-Verified

Methods program.

This research has been funded by the

U. S. Environmental protection Agency

through its Office of Research and Development (ORD) and was conducted at the

Environmental Monitoring Systems Laboratory-Las Vegas. The work is in support

of the Surface Cleanup Issue, EPA Issue

25. This report has been subjected to

ORD’s peer and administrative review and

has been approved as an EPA publication.

Mention of trade names or commercial

products does not constitute endorsement

of recommendation for use.
5

Jeffre C. Johnson (also the EPA Project Officer, see below) and Jeanette M. Van

Emon, are with the Environmental Monitoring Systems Laboratory, Las Vegas, NV

89193-3478.

The complete report, entitled “Development and Evaluation of a Quantitative Enzyme

Linked Immunosorbent Assay (ELISA) for Polychlorinated Biphenyls,” (Order No.

PB95-100038/AS; Cost: $19.50; subject to change) will be available only from:

National Technical Information Service

5285 Port Royal Road

Springfield, VA 22161

Telephone: 703-487-4650

The EPA Project Officer can be contacted at:

Environmental Monitoring Systems Laboratory

U.S. Environmental Protection Agency

Las Vegas, NV 89193-3478

• •

United States

Environmental Protection Agency

Center for Environmental Research Information

Cincinnati, OH 45268

EPA/600/SR-94/112

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