A. |
Intended Use |
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The CAPE
Technologies
High Performance Dioxin/Furan Immunoassay Kit is an Enzyme
Immunoassay (EIA) for analysis of Polychlorinated DibenzoDioxins
and Polychlorinated DibenzoFurans (PCDD/Fs) in prepared sample
extracts. Extracts of fly ash, soil, stack gas, tissue, sediment,
water, or other samples which have been prepared by conventional
extraction methods can be exchanged to methanol for EIA analysis.
Samples can also be prepared by immunoassay specific methods
for analysis by EIA. Please read this kit insert and other related CAPE
Technologies literature carefully to gain maximum understanding of the
capabilities and limitations of the test.
Note: Samples analyzed by EIA must contain either
immunoassay compatible internal standards or no internal standards. Please consult
Technical Note TN-001
for further discussion of internal standards. Refer to other Technical Notes and
Application Notes available from CAPE Technologies for
discussion of technical issues and individual applications.
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B. |
Background Information |
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PCDD/Fs are a family of compounds with the same general
structure. There are 75 dibenzodioxin congeners and 135 dibenzofuran
congeners, containing from 1 to 8 chlorine atoms on the dibenzodioxin
or dibenzofuran nucleus. Only 7 of the 75 PCDD congeners and
10 of the 135 PCDF congeners contain the 2,3,7,8 chlorination
pattern thought to be required for dioxin-like toxicity. Only
these 17 of the 210 total PCDD/F congeners contribute to the
Toxic Equivalency (TEQ) of a sample, which is generally the
critical analytical target. Based on a variety of toxicity
tests, these 17 congeners have been assigned Toxic Equivalency
Factors (TEFs) of 1.0 to 0.001 relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
The PCDD/F congener composition of samples can be highly
variable. Because PCDD/Fs are formed unintentionally by
a variety of chemical and combustion processes, samples
usually contain a mixture of many different congeners. Samples
from different sources often have very different mixtures
of congeners which are consistent within the source. In
most samples, the majority of the PCDD/F mass present does
not contribute significantly to the total sample TEQ. Also,
in most samples, only a few PCDD/F congeners are responsible
for the majority of the TEQ. This immunoassay is designed
to measure sample TEQ by responding to the toxic PCDD/F
congeners in correlation with their TEFs. The variability
of congener composition noted above can cause practical
difficulties in using the immunoassay to produce quantitative
results. Consequently, this kit is designed for screening
analysis of PCDD/F samples at preselected or user determined
levels. Please consult CAPE Technologies
Technical Note TN-004
for further discussion of quantitative use of the kit.
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C.
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Test Principles |
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PCDD/Fs are typically extracted with organic solvents
which are incompatible with the EIA. Before introduction of
the sample into the EIA a solvent exchange is required. PCDD/Fs
have very low volatility and are retained within a thin layer
of detergent after evaporation of the original solvent. Methanol
is added to redissolve the PCDD/Fs and this solution is added
directly to the EIA tubes. It should be noted that the literature
value for solubility of 2,3,7,8-TCDD in methanol is 10 ppm,
which is 1000 times higher than the concentration of the highest
standard recommended for this kit.
During the first EIA incubation, PCDD/Fs are specifically
bound by the anti-dioxin antibodies, which have been immobilized
on the EIA tube surface. After washing away the unbound
material, the bound PCDD/Fs remain and a competitor-HorseRadish
Peroxidase (HRP) conjugate is added. Bound PCDD/Fs occupy
the dioxin binding sites of the antibodies in proportion
to the PCDD/F content of the sample and prevent binding
of the competitor-HRP conjugate. After a short incubation,
unbound conjugate is removed and the test tubes are washed
thoroughly. The amount of conjugate bound by the anti-dioxin
antibody is inversely related to the amount
of PCDD/Fs originally present in the sample.
Finally, a solution of chromogenic HRP substrate and hydrogen
peroxide is added to the test tubes. Color development is
directly proportional to enzyme concentration and inversely
related to the PCDD/F concentration in the original sample.
The test tubes are analyzed using a tube reader or spectrophotometer
to measure the optical density (OD) at 450 nm. The OD values
of unknown samples are compared to the OD values of standards
to determine the level of PCDD/Fs in the samples.
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D.
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Performance
Characteristics
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Sensitivity
and Reproducibility
Standard curve data developed by CAPE
Technologies for the High Performance Dioxin/Furan
Immunoassay Kit are given in Table 1. Response values are
expressed as a percentage of the negative control, which is
200 ppb Triton X-100 in methanol. The detection limit of the
kit is approximately 4 pg 2,3,7,8-TCDD per EIA tube. Results
for samples which are compared to the standards shown in Table
1 must be related to the original sample concentration by
back calculation using the proper dilution and volume factors.
Matrix detection limits will vary according to matrix, sample
size, and dilution factor. Consult the appropriate CAPE
Technologies Application Note for further information.
The data in Table 1 can be used to determine if kit performance
is acceptable. If your results for the standards in Table
1 are not consistently within the percent of negative control
ranges given, contact CAPE Technologies
for assistance.
Table 1:
Sensitivity and Reproducibility of the EIA Standard Curve.
Data are accumulated responses for 2,3,7,8-TCDD standards
in methanol/Triton over ten months. No sample matrix was
present. A total of 41 tests were run in four different
labs. The detection limit, which is approximated by the
I85 or the concentration giving 85% of the negative control
OD, was 3.9±1.4 pg/tube (mean±SD). The midpoint of the curve,
defined as the I50 or the concentration giving 50% of the
negative control OD, was 21.9±7.4 pg/tube (mean±SD).
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pg/EIA tube
(ppb in Standard) |
3.2
(0.32)
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10
(1.0)
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32
(3.2)
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100
(10)
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mean percent
of negative control
(%NC) |
87
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66
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41
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29
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| Standard Deviation (SD) |
6 |
7 |
7 |
6 |
Range of
Mean ±2SD |
74-99 |
51-80 |
27-55 |
17-40 |
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Specificity
The anti-dioxin antibody in this kit binds to different PCDD/F
congeners with different affinities. The specificity of the
test is predominantly for PCDD/Fs which contain 3 to 6 chlorines,
with a strong preference for the 2,3,7,8 chlorinated congeners.
Test specificity roughly parallels the TEF values of the individual
PCDD/F congeners. Crossreactivity data given in the following
table are reactivities relative to 2,3,7,8-TCDD.
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Table
2: Specificity of the EIA. Response curves were
prepared for each congener as noted. The percent crossreactivity
= (((congener I50) ÷ (2,3,7,8-TCDD I50)) x 100). Values are
typically based on 2 to 4 independent curves, each containing
at least 4 concentrations.
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| Toxic Dioxin
Congeners |
Percent
Crossreactivity |
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2,3,7,8-TCDD
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100 |
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1,2,3,7,8-PeCDD
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105 |
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1,2,3,4,7,8-HxCDD
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1.6 |
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1,2,3,6,7,8-HxCDD
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7.9 |
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1,2,3,7,8,9-HxCDD
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39 |
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1,2,3,4,6,7,8-HpCDD
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0.7 |
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OCDD
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<0.001 |
| Toxic Furan Congeners |
Percent
Crossreactivity |
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2,3,7,8-TCDF
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20 |
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1,2,3,7,8-PeCDF
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4.6 |
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2,3,4,7,8-PeCDF
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17 |
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1,2,3,4,7,8-HxCDF
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0.4 |
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1,2,3,6,7,8-HxCDF
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1.0 |
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1,2,3,7,8,9-HxCDF
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3.3 |
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2,3,4,6,7,8-HxCDF
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4.9 |
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1,2,3,4,6,7,8-HpCDF
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0.02 |
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1,2,3,4,7,8,9-HpCDF
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0.9 |
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OCDF
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<0.001 |
| Other PCDD/F
Congeners |
Percent
Crossreactivity |
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2,3-dichlorodibenzo-p-dioxin
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0.13 |
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2,7-dichlorodibenzo-p-dioxin
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0.003 |
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2,3-dichlorodibenzofuran
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0.02 |
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2,7-dichlorodibenzofuran
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<0.002 |
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2,3,7-trichlorodibenzo-p-dioxin
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24 |
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2,3,8-trichlorodibenzofuran
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0.26 |
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1,2,3,4-TCDD
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<0.001 |
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1,2,3,4-TCDF
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<0.001 |
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1,3,6,8-TCDD
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0.05 |
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1,3,6,8-TCDF
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0.007 |
| PolyChlorinated
Biphenyls |
Percent
Crossreactivity |
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3,3',4,4' (PCB 77)
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0.4 |
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3,3',4,4',5 (PCB 126)
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0.5 |
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2,2',4,4',5 (PCB 153)
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<0.1 |
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3,3',4,4',5,5' (PCB 169)
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<0.1 |
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Aroclor 1254
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<0.1 |
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