United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas, NV 89193-3478
Research and Development
EPA/600/SR-94/112 September 1994
EPA Project Summary
Development and Evaluation of a
Quantitative Enzyme Linked
Immunosorbent Assay (ELISA)
for Polychlorinated Biphenyls
Jeffre C. Johnson and Jeanette M. Van Emon
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 u.g/
Kg for Aroclors 1248 and 1242, respec-
tively. The ELISA was characterized for
potential cross-reactivity with 37 struc-
turally related chlorinated benzenes,
anisoles, and phenols which might po-
tentially be present as co-pollutants in
environmental samples. Cross-reactiv-
ity 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 chro-
matography (GC) method for PCBs. Ex-
traction efficiency was found to be
greater than 90%, as determined by ex-
traction of carbon-14 radiolabeled
tetrachloro-biphenyl.
Three sets of real-world samples, ob-
tained from Superfund sites and the
EPA National Enforcement and Investi-
gation Center (NEIC) were analyzed us-
ing the quantitative PCB plate ELISA.
In addition, all samples were analyzed
by outside confirmatory laboratories
using standard EPA GC methods. Sub-
sets of the samples were also extracted
using supercritical fluid extraction and
methanolic Soxhlet extraction. The re-
sults 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 de-
tection and quantitation device when
coupled to an efficient extraction pro-
cedure.
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 devel-
opmental stage are described, along with
accompanying performance characteristic
data. In addition, the analysis of three
sets of real-world soil samples, represent-
ing a wide variety of matrix challenges, is
reported.
After the initial detection of polychlori-
nated biphenyls (PCBs) in the environ-
ment in 1966, mounting evidence led the
U.S. Environmental Protection Agency
(EPA) to classify them as suspected hu-
man carcinogens, due in part to their low
rate of degradation, their tendency to
bioaccumulate, and their carcinogenic na-
ture. 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, ana-
lytical method development resulted in the
codification of a number of standard meth-
ods 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
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Soxhlet extraction, followed by gas chro-
matography (GC) for quantitation of PCBs.
Several factors have generated increas-
ing 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-awak-
ened the controversy surrounding the ac-
tual carcinogenicity and toxicity of PCBs.
In an effort to expand the array of tools
available for PCS research, immunochemi-
cally-based methods are being looked at
to provide data under appropriate circum-
stances.
As a result of the Superfund Amend-
ments and Reauthorization Act of 1986
(SARA), site monitoring and assessment
will represent a large and possibly rising
financial burden directly related to the col-
lection, transport, and analysis of PCS
containing samples. ELISA based meth-
ods have been shown to offer the poten-
tial for data outputs which are complimen-
tary and in some cases comparable to
established methods at a significantly lower
cost on a per analysis basis. ELISA meth-
ods 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, un-
der the Superfund Innovative Technology
Evaluation (SITE) Program, officially man-
dated the use of low cost, rugged, field-
portable 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 analyti-
cal 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 GC-
based methods for many applications, such
as site characterization, mapping concen-
tration 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 PCS 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 regu-
latory activities. Based on the Aroclors
which contaminated these real-world
samples the bulk of data are focused on
Aroclors 1242 and 1248. Initial character-
ization data show that this assay could be
used for Aroclors 1254 and 1260 equally
well with similar performance characteris-
tics. The remainder of the discussion cen-
ters around Aroclors 1242 and 1248.
The assay described in the current study
is intended for the analysis of PCS con-
tamination in solid matrices such as soil,
sediment, clays and paper pulp, and thus
the samples required extraction prior to
analysis. The methanol-based shake ex-
traction procedure employed during the
present study was chosen for its simplic-
ity, and was based on extraction proce-
dures common to a number of field meth-
ods. Preliminary extraction studies, with a
wide variety of matrices, using a radio-
labeled 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 materi-
als (SRMs), followed by quantitation with
the plate ELISA provided an indirect mea-
sure of extraction efficiency. ELISA re-
sults for Aroclor 1248 SRMs suggest ex-
traction efficiencies greater than 90%, while
for Aroclor 1242, employing 5 PCS levels,
efficiencies ranging from 53% to 91% were
observed. In all cases for the 1242 SRMs,
the reported value is within the EPA de-
fined advisory range as specified by SW-
846 Method 8080/81.
The assay had detection limits of 1.31
ng/mL for Aroclor 1248 with a o of 0.9 ng/
mL and a detection limit of 1.6 ng/mL for
Aroclor 1242 with a o value of 0.61 ng/
mL. The detection limit in soil (based on a
5 gram sample) is 9.0 ng/g, o = 6.0 ng/g
for Aroclor 1248 and 10.5 ng/g, o = 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, correspond-
ing 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 |ig/mL PCBs require ap-
propriate dilution to bring the PCS con-
centration into the working range of the
assay.
The assay provided the long-term re-
producibility required for use as a quanti-
tative 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 PCS
level, RSD's ranged from 30% to 5%.
The quantitative PCS 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 mea-
surement of PCS concentrations. The as-
say exhibited selectivity for PCBs which
will allow the plate ELISA to be used in
the presence of a wide range of com-
monly occurring chlorinated anisoles, ben-
zenes and phenol co-pollutants. The 37
compounds which were studied in the pre-
liminary 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 er-
rors, sample preparation differences, and
inter-lab variation, even before variability
is introduced by true inter-method differ-
ences. Comparison of the quantitative PCS
plate ELISA soil sample results with re-
sults 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 lim-
ited 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 com-
paring 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 char-
acteristics when applied to oily clay
samples or sediments.
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ELISA Analysis of Kansas City
Samples
ELISA analysis of Aroclor 1248 con-
taminated 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 labo-
ratory 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 sug-
gest that this is not likely to be the case.
One undesirable alternative would be run-
ning enough replicates of each sample to
ensure the validity of this assumption. This
degree of rigor is possible with immu-
noassay, however, given the time and ex-
pense of the CLP analyses, this is not
practical.
Another problem is the distinction (or
lack thereof) between preparative and de-
terminative 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 el-
evates 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 accu-
rately measure PCB concentrations. The
second hypothesis can be ruled out as
well. Data collected during parallelism stud-
ies 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 extrac-
tion procedures employing methanol would
work better than hexane/acetone extrac-
tions 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 methanol-
based shake extraction used for the ELISA
might offset the gain realized from substi-
tution of methanol. Results for the SFE
extracts of the Kansas City samples illus-
trate 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 metha-
nol shake procedure were not (t = 2.118,
p = 0.046).
The fourth hypothesis cannot be ruled
out easily either. If there are cross-react-
ing compounds, they are not commonly
occuring chlorobenzenes, phenols, or
anisoles. One possibility is the presence
of PCB metabolites, such as polychlori-
nated biphenylols, which would not be de-
tected 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 immunoas-
say 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 ob-
tain a quantitative PCB result of 0.1 mg/
Kg, + 0.1 may have great value when the
option is either GC analysis or a semi-
quantitative "less than 5 mg/Kg" result ob-
tained through use of a commercial, semi-
quantitative ELISA. The quantitative PCB
plate ELISA allows for the measurement
of PCB concentration in a way which pro-
vides more information than the semi-
quantitative ELISAs currently available
commercially, while using an assay pro-
cedure 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 essen-
tially 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 re-
sults overlap one another within the 95%
confidence intervals of the respective
methods. For the high level samples,
ELISA and Method 8081 results over-
lapped 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 ex-
tracts. Again, parallelism studies and spike
recovery data demonstrated that the quan-
titative PCB plate ELISA, as applied to
the Allied Paper/Portage Creek/Kalamazoo
River samples, showed high accuracy and
no assay degradation due to matrix arti-
facts. Thus, the potential utility of the quan-
titative PCB plate ELISA as a determina-
tive 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 differentiat-
ing the sample preparation from the deter-
minative step itself. The ELISA results for
the simple "20 minute methanolic shake"
extracts and the ELISA results for the
methanolic Soxhlet extracts are very dif-
ferent. Clearly, only the extraction effi-
ciency plays a significant role in altering
the ELISA results.
The fact that the ELISA results for the
methanolic Soxhlet extracts are conver-
gent 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 de-
terminative step, provided comparable data
to the GC, for these environmental
samples, provided that appropriate extrac-
tion procedures were used.
ELISA Analysis of NEIC Samples
The results from ELISA analysis of
samples obtained from the EPA Enforce-
ment and Investigation Center (NEIC) il-
lustrate a number of points. Analysis of
several serial dilutions of the sample ex-
tracts demonstrated that the assay was
not subject to performance degradation
due to matrix artifacts. The ELISA results
were generally higher than the correspond-
ing GC results, with an average RPD of
37%.
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The results for this data set raise the
issue of calibration, an issue which is uni-
versal 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 mix-
ture. In addition, the PCB levels reported
by NEIC most likely have a built in bias,
due to analyst judgement calls on assign-
ing 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 con-
tain 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 eas-
ily performed method bridging the perfor-
mance gap between GC methods and
semi-quantitative ELISA.
The real-world data indicate that extrac-
tion 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, be-
cause 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 it-
self.
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 analy-
sis, 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 mea-
surements on two distinct soil extracts.
The two soil extracts are very likely differ-
ent 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 sce-
nario would entail re-extraction of the Kan-
sas City and/or Allied Paper samples, fol-
lowed 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 use-
ful data for certain applications. The indi-
rect 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 origi-
nal 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 num-
bers of samples, and in addition, it could
allow for the carrying out of extensive
parallelism studies on a routine basis. Ex-
tensive quality assurance of the ELISA
data output could thus be ensured.
A note of caution is raised with respect
to simple or abbreviated extraction proce-
dures. Typically, most of the commercially
available semi-quantitative ELISAs for soil
screening rely upon "quick shake" extrac-
tion procedures, enabling extreme stream-
lining of the entire ELISA based proce-
dure. The experiences noted in the cur-
rent 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 accu-
racy and precision when coupled with ap-
propriate sample preparation procedures.
Further utilization should include coupling
the quantitative PCB plate ELISA with ef-
ficient and potentially fieldable extraction
methods, such as supercritical fluid ex-
traction. Additionally, the quantitative PCB
plate ELISA could be coupled with rapid
Soxtec™ type extraction procedures, po-
tentially in mobile laboratories, enabling
rapid, relatively non labor-intensive mea-
surement of PCBs. This would be an ana-
lytical scheme of high utility, and accep-
tance, as use of Soxtec type extraction
procedures for PCBs already has prece-
dence in such methods as EPA SW-846
Method 3541, Automated Soxhlet Extrac-
tion.
Interest in the application of the quanti-
tative PCB plate ELISA has been gener-
ated within a number diverse groups, such
as the EPA Great Lakes National Pro-
gram Office and the Fish and Wildlife Ser-
vice, National Fisheries Contaminant Re-
search 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 Ana-
lytical Chemists (AOAC) Peer-Verified
Methods program.
This research has been funded by the
U. S. Environmental protection Agency
through its Office of Research and Devel-
opment (ORD) and was conducted at the
Environmental Monitoring Systems Labo-
ratory-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 publica-
tion.
Mention of trade names or commercial
products does not constitute endorsement
of recommendation for use.
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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
Official Business
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