14,15-EET/DHET ELISA kit

14,15-EET/DHET ELISA kit
Catalog Number: DH2/DH12/DH22
Store at -20°C.
FOR RESEARCH USE ONLY
V.05262010
Introduction
It is well known that arachidonic acid (AA) will be converted to EET by P450 arachidonic acid epoxygenase (AA
epoxygenase) and EET will be converted to DHET by soluble epoxide hydrolase (sEH) in vivo. Cytochrome P450
2J2 (CYP2J2) is a predominant human AA epoxygenase that produces all four EETs. In human carcinoma cells,
rAAV-mediated over expression of CYP2J2 resulted in a marked increase in 14,15-DHET level in cell plasma,
whereas rAAV-anti2J2-mediated silence of CYP2J2 expression significantly decreased its production (1). Out
EET/DHET kit can be used to measure EET levels in cultured cells which express sEH (1).
DH2, DH12, or DH22 to be used for 14,15-EET/DHET measurement is the same kit as DH1, which is used for the
measurement of 14,15-DHET. The only difference with DH2, DH12, and DH22 compared with DH1 is the sample
preparation step. Instructions are provided as to the proper isolation and purification in the following pages.
Storage and Stability
This kit will obtain optimal results if all of the components are stored at the proper temperature prior to use. Items
should be stored at the designated temperatures upon receipt of this kit.
All components are stored below -20°C and should not be re-frozen and thawed more than necessary.
Materials Provided
Part
Number
1
2
3
4
5
6
7
Item
Description
Quantity
14,15-DHET ELISA
Plate
14,15-DHET Standard
(2 µL)
14,15-DHET-HRP
Conjugates
(12 µL)
Sample Dilution Buffer
(25 mL)
HRP Buffer
(15 mL)
Wash Buffer Solution
(25 mL)
TMB Substrate
(24 mL)
Solid 96-well plate coated with anti-14,15-DHET antibody in
each well
1
Stock standard at a concentration of 1 mg/mL
1
1000 X concentrated solution
1
10 X solution of Tris-buffered saline with preservatives
1
1 X solution of Tris-buffered saline with preservatives
1
10 X solution of Tris-buffered saline with detergents and
preservatives
1
A solution of TMB (tetra methyl benzadine)
1
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Additional Required Materials (Not Provided)
Plate reader with a 450 nm filter
An 8-channel adjustable pipetter and an adjustable pipetter
Storage bottles
Costar® cluster tubes (1.2 mL) and microcentrifuge tubes
Deionized water
Precautions
Please read all instructions carefully before beginning the assay.
The reagents in this kit have been tested and formulated to perform optimally. This kit may not perform
correctly if any of the reagents are replaced or any of the procedures are modified.
This kit is intended for research use only and is not to be used as a diagnostic.
Procedural Notes
Remove all of the reagents required, including the TMB, and allow them to equilibrate to room temperature
before proceeding with the assay.
It is necessary to thoroughly mix the concentrated buffer solutions. A stir bar is contained within each buffer
solution.
Sample Preparations
EET+DHET can be measured after chemically changing EET to DHET. However, if the EET in cells or in blood is
changed to DHET by abundantly expressed soluble epoxide hydrolase, measurement of DHET without chemically
changing EET to DHET is suitable.
For example, when 14,15-DHET levels were measured in urine samples obtained from Spontaneously Hypertensive
rats, 14,15-DHET levels in the urine were measured without changing EET to DHET. High 14,15-DHET levels
were indicative of increased soluble epoxide hydrolase activity of the rat (thus soluble epoxide hydrolase-dependent
hypertension).
However, when P450 2C23 activity of the rat microsomes was measured, the rat microsomes were incubated with
arachidonic acid (substrate of P450 2C23) and, then, EET + DHET levels in the reaction mixture were measured
after acid hydrolysis of EET to DHET, which was indicative of P450 2C23 activity.
There are three different protocols which can be used to convert EET into DHET for measurement using the
competitive ELISA kit. For optimal results please choose the protocol which fits your sample best.
Protocol #1: EET formation activity measurement
1. Collect and homogenize and/or sonicate the cells using a solution containing a final concentration of 0.1
mM TPP (triphenylphosphine). TPP is an antioxidant, which looks like precipitate in samples because it
does not easily dissolve. Before using the stored samples with TPP, spin to separate the TPP from the
samples.
2. Acidify the whole homogenized cells with acetic acid to a pH of approximately 3-4. Measure using
standard pH paper. (Be careful when changing pH by adding 1 µL of acetic acid at a time.)
3. Extract with ethyl acetate. Add an equal volume of ethyl acetate to the homogenized cells, and vortex
thoroughly. Transfer the upper organic phase into a fresh clean tube after centrifugation. Then add another
equal volume of ethyl acetate to the homogenized cells and repeat the extraction two more times.
4. Evaporate the pooled ethyl acetate until all is dried up under argon gas.
5. Add 20 µL of ethanol or N, N-dimethyl-formamide (DMF) to dissolve the dried up residue for
reconstitution. Add 0.5 mL 1x Sample Dilution Buffer (provided in the kit) to make a solution. Load 100
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6.
µL into each well in triplicate on the ELISA plate. (Note: We recommend measuring a different dilution of
the sample in an attempt to fit the results to the standard curve. e.g. Add 50 µL of the rest of the sample
plus 50 µL 1x Sample Dilution Buffer to three wells plus add 10 µL of the rest of the sample plus 90 µL of
1x Sample Dilution Buffer to three wells.)
Perform the ELISA for 14,15-DHET (according to the instructions of the manufacturer).
Protocol #2: Free EET + DHET formation activity measurement
1. Biological samples have to be collected in TPP (triphenylphosphine) with a final concentration of 0.1 mM.
TPP is an antioxidant, which looks like precipitate in samples because it does not easily dissolve. Before
using the stored samples with TPP, spin to separate the TPP from the samples.
2. Acidify the samples with acetic acid to a pH of approximately 3-4. After acidification, the samples are
extracted three times with ethyl acetate. For each extraction, add an equal volume of ethyl acetate to the
sample, vortex thoroughly, spin down, and collect the organic phase. After extracting three times from the
same sample, pool the collected organic phases (ethyl acetate) and evaporate under argon gas.
3. Dissolve the above dried up residue in 20 µL of ethanol then add 20 µL of acetic acid to make the pH
approximately 3-4. In the acidic conditions EET is hydrolyzed to DHET. The reaction usually takes 12 h
at 45°C or 18 h (overnight) at room temperature. The reaction vial has to be flushed with argon and kept
under an argon blanket. (An argon blanket is like a pouch to keep an argon gas flow during the hydrolysis.)
If an argon blanket is not available at your place, you can add clean powdered dry ice to get rid of residual
oxygen.
4. After the reaction, add 1.5X water to the sample and extract the sample three times with equal volume of
ethyl acetate (vortex well, spin down and collect the organic phase). After three times of extraction, pool
all the organic phase (ethyl acetate) together and evaporate under argon or nitrogen.
5. For ELISA assay, dissolve the sediment in 20 uL of ethanol or DMF (vortex thourghly), then add 130 uL
of 1x Sample Dilution Buffer to make stock solution. The stock sample solution can be diluted in a proper
range of concentration for ELISA test. Check the final pH (should be pH 7.4).
6. Use the 14,15-DHET ELISA kit to measure DHET, which includes DHET converted from EET. At the
same time, measure the DHET level without hydrolysis of EET in the same sample. Subtract that value
from the EET + DHET level and you will obtain the EET level in the sample.
Protocol #3: Free and esterified EET + DHET formation activity measurement
1. Biological samples have to be collected in TPP (triphenylphosphine) with a final concentration of 0.1 mM.
TPP is an antioxidant, which looks like precipitate in samples because it does not easily dissolve. Before
using the stored samples with TPP, spin to separate the TPP from the samples.
2. Acidify the samples with acetic acid to a pH of approximately 3-4. After acidification, the samples are
extracted three times with ethyl acetate. For each extraction, add an equal volume of ethyl acetate to the
sample, vortex thoroughly, spin down, and collect the organic phase. After extracting three times from the
same sample, pool the collected organic phases (ethyl acetate) together and evaporate under argon gas.
3. To cleave the esterified eicosanoids, 2 mL of 20% KOH was added and mixed very well. The mixture was
incubated at 50°C for one hour. [Prepare a 20% KOH solution from 1 mL 2M KOH and 4 mL methanol
(final concentration KOH = 0.4 N)].
4. Dilute 2 mL of the aqueous solution with 3 mL of H2O. Adjust the pH using 20% formic acid to pH~5.
Add ethyl acetate (1 part aqueous solution + 1 part ethyl acetate), vortex thoroughly, and centrifuge at 2000
rpm for ten minutes at 22ºC. Repeat the procedure twice more using an equal volume of ethyl acetate per
sample. Pool all the organic phase (ethyl acetate) together and evaporate under argon gas.
5. Dissolve the dried residue in a minimal amount of ethanol (~20 uL), add 20 µL of acetic acid to make a pH
of approximately 3-4. In the acidic conditions EET is hydrolyzed to DHET. The reaction usually takes 12
h at 45°C or 18 h (overnight) at room temperature. The reaction vial has to be flushed with argon and kept
under an argon blanket. (An argon blanket is like a pouch to keep an argon gas flow during the hydrolysis.)
If an argon blanket is not available at your place, you can add clean powdered dry ice to get rid of residual
oxygen.
6. After reaction, add 1.5x water to the sample and extract the sample three times with equal volume of ethyl
acetate. For each extraction, vortex thoroughly and spin down and collect the organic phase. After three
times of extraction, pool all the organic phase (ethyl acetate) together and evaporate under argon.
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7.
8.
For ELISA, dissolve the sediment in 20 uL of ethanol or DMF (vortex thourghly), then add 130 uL of 1x
Sample Dilution Buffer to make stock solution. The stock sample solution can be diluted in a proper range
of concentration for ELISA test. Check the final pH (should be pH 7.4).
Use the 14,15-DHET ELISA kit to measure DHET, which includes DHET converted from EET. At the
same time, measure the DHET level without hydrolysis of EET in the same sample. Subtract that value
from the EET + DHET level and you will obtain the EET level in the sample.
References
1.
2.
3.
Cancer Res. 2005; 65:4707-15.
Circulation. 2004; 110:2132
Letters in Drug Design & Discovery. 2005; 2:239, etc.
Assay Preparations
The solid 96-well plate and TMB solution are provided ready to use. The preparations of other assay reagents are
detailed below.
Wash Buffer: Mix the solution with a stir bar, applying low heat until a clear colorless solution is obtained. Dilute
the entire contents of the Wash Buffer Concentrate (25 mL) with 225 mL of deionized water to yield a final volume
of 250 mL of 1 X Wash Buffer. This can then be refrigerated for the entire life of the kit.
HRP Conjugate: Dilute 1 vial of the 14,15-DHET-HRP conjugate (0.012 mL) with 12.00 mL of 1 X HRP buffer.
One vial makes enough conjugate for one plate. The conjugate must be used the same day and should not be stored
for later use.
Standards: Label 5 microtubes as Standard 1 through Standard 5. Dilute the entire contents of Sample Dilution
Stock buffer (25 mL) with 225 mL deionized water to yield a final volume of 250 mL of 1 X Sample Dilution
Buffer. Add 0.9 mL of the Sample Dilution Buffer to the microtubes for Standards 1 to 5. Spin down the enclosed
14,15-DHET standard vial (2 µL, filled with inert gas) and add 1.998 mL of Sample Dilution Buffer to obtain 2 mL
of solution. Label this Standard 6. Add 0.1 mL of the Standard 6 to the microtube labeled Standard 5 and mix
thoroughly. Next, add 0.1 mL of Standard 5 into the microtube labeled Standard 4 and mix thoroughly. Continue to
serially dilute the standards using 1:10 dilutions for the remaining standards.
Samples: Samples can be directly diluted into the 1 X Sample Dilution Buffer if it is in solution. For extracted and
dried samples, it is recommended to dissolve the dried-up samples with a minimal amount of ethanol of N, Ndimethyl-formanmide (DMF, 10 µL to 20 µL) and vortex well. Before ELISA assay, add 100 µL of 1 X Sample
Dilution Buffer to make the stock sample solution ready for quantification with ELISA. The stock sample solution
can be further diluted to a proper range of concentration for ELISA test.
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Performing the Assay
Plate Setup: Each plate must contain a minimum of three blank wells (BL), three maximum binding wells (BO), and
a six point standard curve (S1-S6). Each sample should be assayed in triplicate. A suggested plate format is shown
below:
BL
BL
BL
B0
B0
B0
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
=BL
=BO
=S1—S6
=Samples
Standard Dilutions Table
Standards
No. 6
No. 5
No. 4
No. 3
No. 2
No. 1
Final Concentration (pg/mL)
1,000,000
100,000
10,000
1,000
100
10
Add Sample Dilution Buffer (mL)
1.998
0.9
0.9
0.9
0.9
0.9
Serial Dilutions Procedure
2 µL of stock solution.
Add 0.1 mL of No. 6
Add 0.1 mL of No. 5
Add 0.1 mL of No. 4
Add 0.1 mL of No. 3
Add 0.1 mL of No. 2
Assay Procedure
Step 1: Load 200 microliters of Sample Dilution Buffer into the blank (BL) wells and 100 microliters of
Sample Dilution Buffer into the maximum binding (BO) wells.
Step 2: Load 100 microliters of each of the standards into the appropriate wells.
Step 3: Load 100 microliters of each of the samples into the appropriate wells.
Step 4: Load 100 microliters of the diluted 14,15-DHET-HRP conjugate in the BO wells, the standard wells,
and the sample wells. Do NOT add HRP conjugate into the BL wells.
Step 5: Incubate the plate at room temperature for two hours.
Step 6: Wash the plate three times with 400 microliters of the diluted Wash Buffer per well.
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Step 7: After the last of the three wash cycles pat the plate dry onto some paper toweling.
Step 8: Add 200 microliters of the TMB substrate to all of the wells (including BL wells).
Step 9: Incubate the plate at room temperature for 15-30 minutes.
Step 10: Add 50 micoliters of 2 N sulfuric acid to all of the wells.
Step 11: Read the plate at 450 nm.
Calculating the Results
Most plate readers provide data reduction software that can be used to plot the standard curve and determine the
sample concentrations. If your plate reader does not have this option, then a data reduction program can be used (4
parameter of log-log curve fit).
If you do not have these options, the results can be obtained manually as follows:
1.
2.
3.
4.
5.
6.
Average the absorbance readings from the blanks and subtract that value from each well of the plate to obtain
the corrected readings. (Note: Some plate readers do this automatically. Consult the user manual of your plate
reader.)
Average the corrected absorbance readings from the BO wells. This is your maximum binding.
Calculate the %B/BO for Standard 1 by averaging the corrected absorbance of the two S1 wells, divide the
average by the maximum binding, then multiply by 100. Repeat this formula for the remaining standards.
Plot the %B/BO versus the concentration of 14,15-DHET from the standards using semi-log paper.
Calculate the %B/BO for the samples and determine the concentrations, utilizing the standard curve.
Multiply the concentrations obtained for each of the samples by their corresponding dilution factor.
Typical Results
70
60
% B/B0
50
40
30
20
10
0
1
100
10000
1000000
14,15DHET (pg/mL)
The data shown here is an example of typical results obtained using the
Detroit R & D 14,15-DHET ELISA kit. These results are only a
guideline, and should not be used to determine values from your samples.
The user must run their own standard curve every time.
BL wells
BO wells
= 0.070
= 0.869
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Standard
No. 1
No. 2
No. 3
No. 4
No. 5
No. 6
Concentration
10 pg/mL
100 pg/mL
1,000 pg/mL
10,000 pg/mL
100,000 pg/mL
1,000,000 pg/mL
O.D.
0.540
0.445
0.300
0.094
0.020
0.011
%B/BO
67.6
55.7
37.5
11.8
2.5
1.4
Specificity of anti-14,15-DHET IgG
The specificity of the 14,15-DHET ELISA was investigated using authentic 14,15-DHET and a panel of
eiconsanoids.
14,15-DHET
8,9-DHET
11,12-DHET
14,15-EET
15(s) HETE
8,9-EET
5(s)15(s)DiHETE
11,12-EET
Arachidonic Acid
5,6-DHET
5,6-EET
Thromboxane B2
PGE2
PGF2a
6-keto-PGF1a
100.00 %
3.30 %
3.30 %
1.5 %*
1.00 %
0.40 %
0.20 %
0.05 %
0.05 %
0.02 %
0.02 %
0.02 %
<0.01 %
<0.01 %
<0.01 %
*Recent experiment showed 0.3% cross-reactivity.
Troubleshooting
No color present in standard wells.
The HRP conjugate was not added. Redo the assay and add the conjugate at the proper step.
The HRP conjugate was not incubated for the proper time. Redo the assay and incubate for the proper
time.
No color in any wells, including the TA wells.
The TMB substrate was not added. Add substrate.
The TMB substrate was not incubated for the proper time. Continue incubation until desired color is
reached.
The color is faint.
One or all of the incubation times were cut short. Redo the assay with the proper incubation times.
The TMB substrate was not warmed up to room temperature. Redo the assay making sure all reagents
are at room temperature.
The lab is too cold. Be sure the lab temperature is between 21-27°C and redo the assay.
The background color is very high.
The TMB substrate has been contaminated. Redo the assay with a fresh bottle of substrate.
Scattered O.D. obtained from the sample.
Redo assay using an 8-channel pipetman making sure that 8 channels are equal volume while loading.
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Warranty
Detroit R&D, Inc., makes no warranty of any kind expressed, or implied, including, but not limited to the warranties
of fitness for a particular purpose and merchantability.
Detroit R&D, Inc.
Metro Center for High Technology Bldg. (MCHT)
2727 Second Ave. Suite 4113
Detroit, MI 48201
Phone: 313.961.1606
Fax: 313.963.7130
E-mail: info@detroitrandd.com
www.DetroitRandD.com
2727 Second Ave. Suite 4113 – Detroit, MI 48201 – tel. 313.961.1606 – fax. 313.963.7130 – info@detroitrandd.com – www.detroitRandD.com