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
Penalty for Private Use $300
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         EPA
   PERMIT No. G-35
EPA/600/SR-94/112

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