Procept® - Eichrom

Procept ® Rapid Dioxin Assay
Kit Insert
expertise.commitment.results.
Contents:
A. Materials Provided/Storage Conditions
B. Additional Materials/Equipment Required
C. Background Inforamation
D. Intended Use
E. Instructions for Use of Kit
F. Performance Characteristics
G. Safety
H. Pipetting Tips
I. References
J. Warranty
K. Procept ® Quick Reference
A. Materials Provided/Storage Conditions
1) 12 Capture Strips (Foil Pouch, Store at Room Temperature)
2) 1-96 plate of glass reaction vials (Store at Room Temperature)
3) Assay Buffer (Store at room temperature for up to a week, refrigerate for longer storage)
4) 25x Wash Buffer (Store at room temperature for up to a week, refrigerate for longer storage)
5) 1 vial of Capture Reagent (Red Cap, Store at -20 o C)
6) 1 vial of Primer/Probe (White Cap, Store at -20 o C)
7) 6 vials of Activation Solution (Clear cap, brown solution, Store at -80 o C)
Note: Activation Solution must be kept at -80 o C until use in a -80 o C freezer, with dry ice or with liquid
nitrogen. Thaw activation solution immediately prior to use. Once thawed, activation solution must be
used or discarded. Thawing and re-freezing activation solution will result in decreased performance of the
assay.
B. Additional Materials and Equipment Required
1) Plate Washer (Biotek Elx50 or equivalent)
2) Plate Shaker (Heidolph Titramax 100 or equivalent)
3) Real-time PCR
4) Calibrated delivery pipets (1-20 µL, 20-200 µL and 100-1000 µL)
5) Barrier pipet tips
6) Optically Clear Adhesive Film (Applied Biosystems no. 4311971)
7) Optical Compression Pads (Applied Biosystems no. 4132639)
8) Deionized water
9) DNA/RNase free water
10) Real-time PCR Master mix
11) 2,3,7,8-Tetrachlorodibenzo-p-dioxin standard
C. Background Information
Polychlorinated dibenzo-p-dioxins and furans (PCDD/F) are a class of environmentally persistent organic
pollutants. Most of the toxic and biological effects of PCDD/Fs have been attributed to interaction with the
aryl-hydrocarbon receptor (AhR), a cytosolic receptor protein found in most vertebrate tissues. The world
health organization (WHO) has assessed the relative toxicity of seventeen PCDD/F congeners (a
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PCDD/F compound containing a specific number and pattern of chlorine substituents) based on the ability
of the congener to bind to the AhR, elicit AhR-mediated biochemical and toxic responses and be
persistent and accumulate in the food chain. These seventeen PCDD/F congeners have been assigned
toxicity equivalent factors (TEFs) relative to the most toxic congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
In standard gas chromatography mass spectrometry (GC-MS) determinations, toxicity equivalent
quotients (TEQ) of PCDD/F containing samples are determined by taking the sum of the concentrations
of each PCDD/F congener multiplied by its TEF.
The PROCEPT ® assay utilizes the affinity of the aryl hydrocarbon receptor for PCDD/F
compounds, forming complexes composed of PCDD/F, the AhR, an aryl hydrocarbon nuclear translocator
protein (ARNT) and a specific DNA response element (DRE). This complex is then bound to a plastic strip
coated with a polyclonal antibody specific for the c-terminus of the ARNT. Excess AhR, ARNT and DNA
are washed away and the amount of DNA is amplified and measured using real-time polymerase chain
reaction (PCR). The output of the PCR instrument is threshold cycle (Ct). The Ct is the number of PCR
temperature cycles at which the measured fluorescence for a given sample/standard exceeds a threshold
value. By comparing the Ct of an unknown sample to the Ct values of a standard curve generated from 5-
7 concentrations of 2,3,7,8-TCDD, the TEQ of the sample can be determined.
The degree of interaction of individual PCDD/F congeners with the AhR is proportional to the TEF
established by the WHO, therefore, the amount of DNA measured by the PCR is proportional to the
PCDD/F TEQ of the sample. However, the PROCEPT ® assay does not provide information on the
amount of each individual PCDD/F congener.
Several other classes of aryl hydrocarbon compounds also interact with the AhR, including
polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and brominated and mixed
brominated/chlorinated dibenzo-p-dioxins and furans. Therefore, in order to measure PCDD/Fs using the
AhR-PCR assay, a sample preparation method capable of separating PCDD/Fs from other AhR active
compounds is necessary. A complete list of cross-reactivity values and appropriate sample preparation
methods can be found in literature available at www.eichrom.com/dioxin/products.
D. Intended Use
The Procept Rapid Dioxin Assay is an Aryl-hydrocarbon (AhR) based polymerase chain reaction (PCR)
assay for the measurement of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) in prepared
sample extracts. Samples should be prepared for measurement by the Procept Assay using an approved
method for isolating PCDD/F from the sample matrix (see methods available from Eichrom Technologies
LLC at www.eichrom.com/dioxin/products). Consult the notes listed below in this package insert and
literature available at the Eichrom Technologies website for limitations of this method.
Note: The Procept Rapid Dioxin Assay measures total PCDD/F response which correlates with the TEQ
of the sample. The Procept Assay will not provide information on the concentration of individual PCDD/F
congeners present in the sample.
Note: The AhR used in this method binds to PCDD/F and similar molecules based on structure, not mass.
Therefore, 13 Carbon- or other stable isotope labeled standards are detected to the same degree as native
compounds. These labeled standards cannot be used as internal standards with this method.
Note: The Procept Rapid Dioxin Assay is designed for screening of samples according to their TEQ by
responding to PCDD/F congeners in proportion to their concentration and TEF value. However, the
response factors for individual PCDD/F congeners on the AhR-PCR assay are not identical to the TEF
values assigned by the world health organization (WHO). Therefore, variation in the accuracy among
samples may occur solely because of variability in congener composition. Quantitative interpretation of
the data may be possible in certain situations. However, confirmation of positive identification of PCDD/F
contamination and a portion of negative samples by GC-MS is strongly recommended.
E. Instructions for Use of Kit
1. Sample Extraction and Purification
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Sample extraction and purification techniques will vary with sample matrix. Please consult the
Eichrom website (www.eichrom.com/dioxin/products) for appropriate methods for the matrix of
interest.
2. Pre-analysis Preparation
2.1. Preparation of 2,3,7,8-TCDD standards by serial dilution.
2.1.1. The 2,3,7,8-TCDD standard is received as a 5 ug/mL solution in nonane. Prepare
a 200 ng/mL dilution in a 4 mL glass vial by dissolving 80 uL of the standard
in 1920 uL of heptane. Measure volumes using automatic delivery pipets and
barrier pipet tips.
2.1.2. Prepare a 5000 pg/mL dilution in a 4 mL glass vial by dissolving 50 uL of the 200
ng/mL dilution in 1950 uL of heptane.
2.1.3. Prepare a series of 2x serial dilutions of the 5000 pg/mL standard (5000, 2500,
1250, 615, 313, 156 and 78 pg/mL).
2.1.3.1. Label six 4 mL glass vials
a. (2500, 1250, 625, 313, 156 or 78 pg/mL TCDD)
b. Date Prepared
2.1.3.2. Add 1 mL of heptane to each 4 mL glass vial.
2.1.3.3. To the 2500 pg/mL vial, add 1 mL of the 5000 pg/mL standard
using a 100-1000 uL pipet and barrier pipet tip.
2.1.3.4. Seal the 2500 pg/mL vial with a PTFE lined cap a mix thoroughly
using a vortex mixer.
2.1.3.5. Using the 100-1000 uL pipet and a fresh barrier pipet tip, add 1
mL of the 2500 pg/mL standard to the 1250 pg/mL vial. Seal the
vial with a PTFE lined cap and mix thoroughly using a vortex
mixer.
2.1.3.6. Complete the series of 2x serial dilutions as described for the
2500 and 1250 pg/mL standards, using a fresh barrier pipet tip
for each dilution.
2.1.3.7. Prepare an assay blank standard by adding 1 mL of heptane to a
4 mL glass vial.
2.1.3.8. Seal the standard vials securely with PTFE caps and store the
standards out of direct sun light.
3. Sample Analysis
3.1 Preparation of Capture Strips
3.1.1. The wash solution is prepared by diluting 40 mL of the wash solution concentrate to 1
L with deionized water. The wash solution is then placed into a glass flask and used to
prime the plate washer (BioTek ELx50, new buffer prime).
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3.1.2. The desired number of capture strips are then placed into the orange rack and
washed using the plate washer (3x wash) to remove the protective coating.
3.1.3. The capture reagent is thawed and diluted in a glass test tube with the assay
buffer (40 µL of capture reagent to 600 µL of assay buffer per capture strip).
3.1.4. Using an eight-channel automatic delivery pipette and 100 µL barrier pipette tips,
50 µL of the diluted capture reagent is added to each well of the capture strips.
3.1.5. The capture strips are then placed on the plate shaker (Heidolph Titramax 1000 or
equivalent, speed set at 900) for 60 to 90 minutes.
3.2. Reaction of Samples and Standards with Ah-Receptor (Performed while capture strips are on
the plate shaker)
3.2.1. For each well in the capture strips used, a glass reaction vial is charged with 25 µL of
assay buffer (8-channel automatic delivery pipette and 100 µL barrier pipette tips).
3.2.2. 5-10 µL of the purified sample extract or standard is added to each glass vial (0.1
to 20 µL automatic delivery pipette and barrier pipette tips).
3.2.3. For every two capture strips used, one vial of the activation solution (stored at -
80 o C or in a liquid nitrogen dewar) is thawed and 25 µL is added to each glass
reaction vial (8-channel automatic delivery pipette and 100 µL barrier pipette tips).
3.2.4. The rack of glass reaction vials is placed on the plate shaker for 60 minutes.
3.3. Addition of reaction mixture to capture strips
3.3.1. After 60 to 90 minutes on the plate shaker, the capture strips are washed using the
plate washer (3x wash) to remove any excess capture reagent.
3.3.2. Using the 8-channel automatic delivery pipette and 100 µL barrier pipette tips,
30 µL of each solution from the glass reaction vials is added to each corresponding
capture strip.
3.3.3. The capture strips are placed on the plate shaker for 30 minutes.
3.3.4. Following 30 minutes on the plate shaker, the capture strips are washed using the
plate washer (5x wash). This takes approximately 15 minutes.
3.4 Polymerase Chain Reaction (PCR)
3.4.1. While the capture strips are on the plate washer, the PCR reagents are prepared per
the manufacturers instructions. For each capture strip used, mix 140 µL of DNase free
water, 175 µL of PCR master mix and 35 µL of primer probe solution in a glass test
tube.
3.4.2. When the 5x wash program is complete, using the 8-channel automatic delivery
pipette and 100 µL barrier pipette tips, 40 µL of the PCR reagent is added to each
well of the capture strips.
3.4.3. OPTIONAL STEP - only necessary for newer ABI models, such as 7300, 7500 and
7900ht. Add 20 µL of hot DNA/RNase free water (near boiling, 90-100 o C) to each
well using the 8-channel automatic delivery pipette. Place the strips on the plate
shaker and mix for 2-3 minutes. Add 16 µL of water from each well to a standard
96-well plate. Add 24 µL of PCR reagents, consisting of 5 parts 2x mastermix to 1
part primer/probe solution (vial with white cap) to each well.
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3.4.4. The capture strips (or 96-well plate wells) are sealed using optically clear adhesive
film (Applied Biosystems part no. 4311971).
3.4.5. Two optical cover compression pads are placed on top of the sealed capture
strips, and the capture strips are placed in the PCR instrument.
3.4.6. The quantitative PCR program is run using the following parameters:
Quantification dye:
Reference dye:
Thermal Profile:
FAM
ROX
2 minutes at 50 o C
10 minutes at 95 o C
Cycle between 15 seconds at 95 o C then 60 seconds at
60 o C (40 times)
F. Performance Characteristics
Table 1 depicts data for typical performance of the Procept ® Rapid Dioxin Assay standard curve as well
as acceptable QA criteria. If results of the standard curve are not consistently within the QA criteria in
Table 1, please contact Eichrom Technologies LLC for assistance.
Table 1. Standard Curve Metrics (Ct vs pg/mL 2,3,7,8-TCDD)*
Eichrom
Acceptable
Historical
Range
Ct range 0-5000 5.5 >4.5
R 2 (78-5000) 0.980 >0.97
average SD of replicates 0.20 3σ from the assay blank, is 0.4 pg 2,3,7,8-TCDD per well. Actual achievable method
lower limits of quantitation will depend on several factors including sample size, extract volume and recoveries
of analytes through extraction and clean-up steps.
Table 2 depicts cross-reactivity values for the 17 most toxic PCDD/F congeners on the Procept ® Assay.
For a complete list of compounds with a significant cross-reactivity on the Procept ® Assay, please see
literature available at www.eichrom.com/dioxin/products.
G. Safety
This method does not address all safety issues associated with its use. The laboratory is responsible for
maintaining a safe work environment and a current awareness file of OSHA regulations (or applicable
health and safety regulations) regarding the safe handling of the chemicals listed in this method. A
reference file of material safety data sheets (MSDS) should be available to all personnel involved in these
analyses.
The AhR-PCR assay should only be used by properly trained personnel in an appropriate laboratory
environment. Personnel should wear appropriate personal protective equipment, including safety glasses,
lab coat and gloves.
PCDD/F standards, solutions containing PCDD/Fs and potentially contaminated samples should be
treated as hazardous materials.
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Some test components are stored in freezers (-20 o C and -80 o C). Care should be taken to avoid
direct contact of frozen components with skin.
H. Pipetting Tips
Table 2. Cross-Reactivity on the Procept ® Assay
Procept® Assay
Congener WHO 2005 TEF response factor
2,3,7,8 TCDD 1 1
1,2,3,7,8 PCDD 1 0.6
2,3,4,7,8 PCDF 0.3 0.3
1,2,3,7,8,9 HxCDD 0.1 0.5
1,2,3,4,7,8 HxCDF 0.1 0.4
1,2,3,4,7,8 HxCDD 0.1 0.4
1,2,3,7,8,9 HxCDF 0.1 0.3
1,2,3,6,7,8 HxCDF 0.1 0.2
1,2,3,6,7,8 HxCDD 0.1 0.1
2,3,4,6,7,8 HxCDF 0.1 0.1
2,3,7,8 TCDF 0.1 0.06
1,2,3,7,8 PCDF 0.03 0.1
1,2,3,4,6,7,8 HpCDD 0.01 0.01
1,2,3,4,6,7,8 HpCDF 0.01 0.05
1,2,3,4,7,8,9 HpCDF 0.01 0.02
1,2,3,4,6,7,8,9 OCDD 0.0003 0.000003
1,2,3,4,6,7,8,9 OCDF 0.0003 0.0005
The precision and accuracy of the Procept ® Rapid Dioxin assay can be affected by the pipetting
technique of the user. Below are several pipetting tips for achieving the best possible results with the
Procept ® Rapid Dioxin Assay.
-Be consistent. Have a pipetting routine and keep the routine consistent throughout the samples in
a single assay and between multiple runs of the assay.
-When pipetting solvents with a higher vapor pressure than water (i.e. heptane or hexane), practice
positive displacement pipetting. Draw the solvent into the pipet tip and dispense to the first stop on
the dispensing mechanism (do not depress the dispensing mechanism to the second stop) to prime
the pipettor. Repeat this procedure 2-3 times, then, draw the solvent into the pipette tip a final time
and dispense the solvent into the desired container by depressing to the first stop of the dispensing
mechanism. A small amount of solvent may remain in the pipette tip, but an accurately measured
amount of the solvent will have been dispensed.
-Use a fresh pipette tip for each sample/standard during the assay and for each dilution when
preparing the 2,3,7,8-TCDD standards. This will minimize the possibility of cross-contamination
between samples or standards.
-Use the lid from the box of pipette tips and the pipette tips themselves to keep track of your
position on the 96-well plate. When using a full test kit, it can be difficult to keep track of where each
sample or standard should be added on the 96-well plate. The lid of the pipette tip box can be used
to keep track of the column to which each sample or standard should be added. As the user
progresses through the test and reaches the end of a column, the lid can be moved to the next
column of the 96-well plate. Additionally, the pipette tips are organized in the same format as the
96-well plate. By using a fresh pipette tip for each sample/standard and removing the pipet tips in
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the same order as one adds the samples or standards to the 96-well plate, the user can keep track
of the position in which to add the next sample/standard.
I. References
Links to technical references, application notes, and analytical methods can be found at the Eichrom
Technologies LLC website: http://www.eichrom.com/dioxin/products. Any questions regarding equipment
or reagent compatibility can be directed to Eichrom technical support at 630-963-0320.
J. Warranty
Eichrom’s products are warranted to conform to specifications contained in our ISO 9001:2000 quality
system up to the product expiration date or one year from the date of purchase 1 . Eichrom will accept
return of any of its analytical products that are defective and consequently do not perform the analytical
process they are designed to perform, provided that defective products are free of all biological
contamination.
Eichrom will, at its option, refund, repair or replace defective products with another lot number of the
same product or credit the purchase price against a future product purchase. Return shipments will be at
the shipper’s expense unless otherwise authorized in writing by Eichrom. Please contact Eichrom for
authorization to return defective materials.
The warranties by Eichrom set forth in this kit insert are intended solely for the benefit of the purchaser of
Eichrom’s product. All claims hereunder shall be made by the purchaser. The warranties by Eichrom set
forth above are in lieu of all other warranties, express or implied, which are hereby disclaimed and
excluded by Eichrom, including any warranty or merchantability or fitness for a particular purpose or use
and all obligations or liabilities on the part of Eichrom for damages arising out of or in connection with the
use or performance of products. Eichrom shall not be liable to the purchaser of its products nor to any
third party for any damages in excess of the purchase price of its product, nor for any special,
consequential, exemplary or incidental damages (including lost or anticipated revenues or profits relating
to the same), arising from any claim relating to any product, whether such claim is based on warranty,
contract, tort, (including negligence or strict liability) or otherwise, even if an authorized representative of
Eichrom is advised of the possibility or likelihood of same. Purchasers of Eichrom products acknowledge
and agree that payment by Eichrom or the retention by purchaser of such amount is limited by the
foregoing sentence and shall be the purchaser’s sole and exclusive remedy in exhaustion of all other
remedies at law or in equity and that such remedy shall not be deemed or alleged by purchaser to have
failed of its essential purpose.
The products described in this kit insert are for laboratory or process control use only. They are not
intended for medicinal use. Eichrom assumes no responsibility if these products are used for medicinal
purposes or are misused in any way.
Eichrom strives to always meet our customers’ needs. If at any time you have questions pertaining to the
performance of an Eichrom product, please do not hesitate to call us at 800-422-6693 or 630-963-0320.
1 Eichrom Technologies LLC. warrants the Procept ® rapid dioxin assay for a period not to extend beyond
the expiration date printed on the product or one year from the date of purchase, provided the
components are maintained as instructed in Section A of the Procept ® Rapid Dioxin Assay kit insert.
Eichrom manufactures these products under our ISO 9001:2000 quality assurance system.
Procept, Eichrom and the Eichrom logo are registered trademarks of Eichrom Technologies LLC
©2007 Eichrom Technologies LLC
All rights reserved. Printed in USA
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L. Procept Quick Reference
Platewasher Set Up
1) Dilute 40 mL of PCR wash concentrate with
960 mL of deionized water.
2) Add the wash solution to the reservoir of the
plate washer.
Wash
Buffer
+ H 2 O
3) Prime plate washer with PCR wash solution.
Prepare Capture Strips
1) Wash the capture strips using the “3X Wash”
program.
2) Thaw capture reagent (red cap).
3) In a glass test tube, dilute 40 µL of Capture
Reagent concentrate with 600 µL of Assay
Buffer for each capture strip used.
+
Assay
Buffer
4) Using an 8-channel pipetter, add 50 µL of the
Capture Reagent to each well of the capture
strips.
50 µL
5) Place capture strips on a plateshaker and shake
for 60-90 minutes.

Sample/Activation Solution Reaction
1) Ready a rack with enough glass vials for each
sample and standard to be used in the current
analysis.
Assay
Buffer
2) Add 25 µL of Assay Buffer to each glass vial
using 8-channel pipetter.
25 µL
3) Using a 20 µL pipetter and filter tips, add
5 – 10 µL of sample or standard to each glass
vial.
Cl
Cl
O
Cl
O
Cl
5 -10 µL
4) Thaw one vial of activation solution for each 2
strips used in testing.
Do not allow activation solution to remain at
room temperature for more than 20 minutes
prior to use.
5) Add 25 µL of thawed Activation Solution to
each glass vial.
25 µL
6) Place the rack of glass vials on a plateshaker
and shake for 60 minutes.

Binding Complex on Capture Strip
1) Wash capture strips on the platewasher using
the “3X Wash” program.
2) Using an 8-channel pipetter, add 30 µL of
each reaction to a well of the capture strips.
30 µL
3) Place the rack of capture strips back on the
plateshaker and shake for 30 minutes.
Prepare Capture Strips for PCR amplification
1) Wash capture strips on the platewasher using
the “5x Wash” program. The series of washes
and soaks takes 15 minutes.
2) Thaw primer probe (white cap) and add 40 µL
primer/probe, 160 µL of water (DNase, RNase
free) and 200 µL of Universal Master Mix per
capture strip used to a glass test tube. Mix
gently.
+ Master + H 2 O
Mix
3) Add the primer/probe + Master Mix solution
to a pipetting trough, and using an 8-channel
pipetter, add 40 µL to each well of the capture
strips.
40 µL
4) Seal the wells with an adhesive optical cover,
cover with 2 compression pads and insert the
strips into the thermocylcer of the PCR
instrument.