AN ENZYME-LINKED IMMUNOSORBENT ASSAY (EL1SA)
for Determining Dioxins in Sediment and Soil Samples

Jeanette M. Van Emon1, Jane C Chuang2, Robert A. Lordo2, Mikaela Nichkova3, Shirley J. Gee3, and Bruce D. Hammock3

'U.S. EPA, ORD, NERL, MDAB, Las Vegas, Nevada, 2Battelle Memorial Institute, Columbus, Ohio, 'University of California, Davis

Background

Dioxins are highly toxic environmental contaminants
that have been linked to cancer, liver damage, and
various reproductive and developmental diseases
(1). Environmental contamination by the dioxins
(polychlorinated dibenzo-p-dioxins [PCDDs]) and
the related polychlorinated dibenzofurans (PCDFs)
is of great concern due to their persistence in the
environment and adverse effects on wildlife and
humans.

There are 75 different PCDD and 135 different
PCDF congeners each having its own chemical
and toxic characteristics. The most widely known
congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) which is also the most toxic. The dioxins
usually exist as mixtures complicating the issues of
waste management, toxicity assessment, human
exposure and environmental impact. Total releases
are not the best measure of the actual toxicity of
these compounds because each compound has
its own level of toxicity. Toxic equivalency factors
(TEFs) are used to represent the fraction of TCDD
toxicity exhibited by a congener or congener group. A
Toxicity Equivalence (TEQ) is then derived from the
concentration of each of the toxic congeners in the
mixture.

An early symptom of dioxin exposure is a persistent
but non-fatal skin rash called chloracne.

The analysis of PCDDs and PCDFs is of great
importance for environmental monitoring and
human exposure assessment. Unfortunately, the
analysis of these compounds is complex and
expensive, limiting the number of samples that can
be analyzed in a timely and cost-effective manner.
Gas chromatography with high resolution mass
spectrometry (GC-HRMS) is the reference analytical
method for the dioxins. However, GC-HRMS requires
intensive sample cleanup procedures and expensive
instrumentation. Analysis costs range from $1,500-
S5.000/sample (2). immunochemical methods such
as the enzyme-linked immunosorbent assay (ELISA)
are based on specific antibodies combining with a
target compound or group of compounds. ELISA
methods have been developed for many compounds
of environmental and human health concern providing
improvements in cost, sensitivity and sample
throughput relative to instrumental methods (3).

References

(1)	National Primary Drinking Water Regulations, Technical Fact Sheet on Dioxin
(2,3,7,8-TCDD), ERA 821-B-94-005.

(2)	G. Shan, W.R. Leeman, S.J. Gee, J.R. Sanborn, A.D. Jones, D.P.Y Chang
and B. D. Hammock, Anal. Chim. Acta 444 (2001) 169-178.

(3)	J.M. Van Emon, JOAC, vol. 84, no. 1 (2001) 125-133.

ELISA

An ELISA was developed to detect dioxins in
environmental samples (2). The method uses the
less toxic 2,3,7-trichloro-8-methyl-dibenzo-p-dioxin
(TMDD) as an alternative standard for TCDD. The
ELISA is based on a competitive reaction between
dioxin in the sample and a protein-dioxin conjugate
adsorbed to the sides of a microwell ELISA plate. An
enzyme provides a color reaction to measure the
amount of dioxin in the sample. The ELISA will detect
many of the PCDDs and PCDFs that have a high TEF
value. Study results indicate that the ELISA method
offers improvement in speed, sample throughput and
cost when compared to GC-HRMS.

Objectives

Develop an immunoassay method to substantially
reduce the complexity and cost of dioxin toxic
congener analysis

Determine TCDD equivalent concentrations in real-
world sediment and soil samples by the ELISA

Determine whether the ELISA-derived TCDD
equivalent results are statistically equivalent to the
TEQ results derived from the 17 PCDDs/PCDFs
GC/HRMS generated concentrations using the
World Health Organization TEF values

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ELISA and GC/HRMS
Performance Comparison

Summary Statistics

ELISA, TCDD equivalent pg/g

GC/HRMS, TEQ pg/g

Sample Size3

75

75

Arithmetic Mean

1690

2400

Standard D evi ati on

2430

3690

Geometric Mean

458

408

Minimum

<8b

6.82

25th Percentile

93

60

50th Percentile

493

661

75,h Percentile

2590

3780

Maximum

13900

17000

ELISA AND GC/HRMS Data
Comparison



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Ranking of Samples Based on
Dioxin TEQ Levels Near the
1000 pg/g Cleanup Level

GC/HRMS Analysis

ELISA
Analysis



# samples $ 1000 pg/g

# samples < 1000 pg/g

# samples $ 1000 pg/g

27

5

# samples < 1000 pg/g

6

38

Conclusions

The dioxin TEQ levels by GC/HRMS and the TCDD
equivalents derived by ELISA were highly linearly
correlated suggesting the ELISA data could be
used to indicate dioxin TEQ levels

Sample-specific differences between methods
were not significant at the 0.05 level indicating that
the two methods yield statistically similar outcomes
(<8 to 14000 pg/g by ELISA and 6.8 to 17000 pg/g
by GC/HRMS)

The ELISA method can be used as an alternative
quantitative monitoring tool for determining dioxin
TEQs in contaminated sediment/soil samples
providing a more cost-effective and timely analysis
for site monitoring and exposure related research

Acknowledgement

This work was funded by the U.S. Environmental Protection
Agency (EPA) under contract number GS-10F-00275K. It has
been subjected to Agency review and approved for presentation.
Mention of trade names and commercial products does not
constitute endorsement or recommendation for use. This
research was also funded by grants from the National Institute
of Environmental Health Sciences 5 P42 ES04699 and NIEHS
Center for Environmental Health Sciences P30 ES05707.

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