11,12­DHET ELISA kit
Catalog Number: DH4/DH14/DH24/DH104
Store at ­20°C.
FOR RESEARCH USE ONLY

V. 10232012
Introduction
This competitive ELISA kit is for determination of 11,12­DHET levels in biological samples. The level of 11,12­
DHET or 11,12­DHET epitope has been shown to exhibit correlation with hypertension in rodents (1,2,3). 11,12­
DHET is a representative metabolite of cytosolic epoxide hydrolase­mediated metabolism of EETs, which are
generated by arachidonic acid epoxygenase activity of cytochrome P450’s (4).
This kit can be used for the determination of 11,12­DHET in serum, plasma, cells, and tissues following proper
isolation and purification. Instructions are provided as to the proper isolation and purification in the following pages.
This competitive ELISA kit, based on competition between 11,12­DHET epitope and 11,12­DHET­HRP conjugate
for a limited number of binding sites available from the anti­11,12­DHET antibody, which is coated to the wells of
the 96 well ELISA plate. The conjugate concentration is held as a constant in each well, while the concentration of
the 11,12­DHET is variable, based on the concentration of the sample or standard. Thus the amount of the 11,12­
DHET conjugate which is able to bind to each of the wells is inversely proportional to the concentration of 11,12­
DHETin the standard or sample. The amount of the conjugate which is bound to each well is then determined by theamount of color obtained, when TMB is added. The TMB reacts with the HRP available in the well. With theaddition of sulfuric acid, the blue colored product is converted into a yellow colored product, which can be read on aplate reader at 450 nm.

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 andshould not be re­frozen and thawed more than necessary.

Materials Provided
Part
Number
1
2
3
4
5
6
7

Item

Description

Quantity

11,12­DHET ELISA
Plate
11,12­DHET Standard(2 μL)
11,12­DHET­HRP
Conjugates(12 μL)
Sample Dilution Buffer(25 mL)HRP Buffer(15 mL)
Wash Buffer Solution(25 mL)TMB Substrate(24 mL)

Solid 96­well plate coated with anti­11,12­DHET antibody ineach well

1

Stock standard at a concentration of 1 mg/mL1000 X concentrated solution

1
1

10 X solution of Tris­buffered saline with preservatives

1

1 X solution of Tris­buffered saline with preservatives

1

10 X solution of Tris­buffered saline with detergents andpreservatives

1

A solution of TMB (tetra methyl benzadine)1

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Page 2 of 9

Additional Required Materials (Not Provided)
§
§
§
§
§

Plate reader with a 450 nm filter
An 8­channel 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
There are different protocols for isolating and purifying 11,12­DHET depending on the medium in which it is in.
Listed below are the different protocols. For optimal results follow the appropriate protocol based on the biologicalsample present.

11,12­DHET measurement in cells
1. Collect and homogenize and/or sonicate the cells using a solution containing a final concentration of~0.1 mM TPP (triphenyphosphine, 0.03­0.05 mg/mL). TPP is an antioxidant, which looks like a
precipitate in samples because it does not easily dissolve. Before using the stored samples containing
TPP, spin samples to separate the precipitated TPP from sample solution.
2. Acidify the whole homogenized cells with acetic acid to a pH of approximately 3­4. Measure using
standard pH paper.
3. Extraction with ethyl acetate. Add an equal volume of ethyl acetate to the homogenized cells andvortex very well. Place the upper organic phase into a fresh clean tube after centrifugation. Then add
another equal volume of ethyl acetate to the homogenized cells to start the second­time extraction. It isstrongly recommended that extraction is performed three times.
4. Evaporate the pooled ethyl acetate from the extractions until all has dried up under argon or nitrogengas.
5. Saponification if needed (see below)
6. Add 10 μL to 20 μL ethanol, or N, N­dimethyl­formamide (DMF), to dissolve the dried­up residue
from above step #4. Add 0.5 mL of 1x Sample Dilution Buffer (provided in kit). Load 100 μL in eachwell, in triplicates, on the ELISA plate. (Note: We recommend measuring a different dilution ofsample in attempt to fit the results to the standard curve. e.g., add 3 wells by 50 μL of the rest of
sample plus 50 μL of 1x Sample Dilution Buffer, and 3 wells of 10 μL of the rest of sample and 90 μL
of 1x Sample Dilution Buffer.)
7. Perform the ELISA for 11, 12­DHET (according to the instructions of the manufacturer).

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Page 3 of 9

Saponification (to cleave fatty acid from glycerol backbone):
1. Dissolve dried fatty acids (obtained from 3X ethyl acetate extractions) in 2mL of 20% KOH solution(make working solution: 1 mL of 2 M KOH + 4 mL methanol with final concentration of KOH = 0.4
N).
2. Vortex and incubate for 1 h at 50°C.
3. Add 1.5X H2O to the solution and adjust pH with 20% formic acid to pH ~5.
4. Re­extract the solution with ethyl acetate (1 part aqueous solution + 1 part ethyl acetate) and dry.

11,12­DHET measurement in tissues
1. Homogenize 1 g of tissue, 4 mL of H2O, and 0.01 mg TPP.
2. Acidify the homogenate by adding 8 μL of acetic acid to each homogenate.
3. Extract with an equal amount of ethyl acetate, vortex thoroughly, spin down, and collect the organic
phase. Repeat this extraction twice more and combine all of the organic phases.
4. Dry the organic substance with argon or nitrogen gas.
5. Saponification if needed (see 11,12­DHET measurement in cells)
6. Dissolve the dried residue from above step #4 with ethanol or DMF. (Add approximately 20 μL of
ethanol or DMF to reconstitute the dried­up residue).
7. Dilute further with 1x Sample Dilution Buffer. Add approximately 0.5 mL of 1x Sample Dilution
Buffer and centrifuge at 10,000 rpm for five minutes at room temperature. The supernatant will be
used for ELISA.
8. Perform the ELISA for 11, 12­DHET (according to the instructions of the manufacturer).

11,12­DHET measurement in plasma or serum
Below are two protocols for measuring 11,12­DHET in plasma or serum using starting volumes of 1.0 or 1.8 mL.
Starting volumes below 1.0 mL can also be used (i.e. for mouse studies) for measuring 11,12­DHET by making
appropriate adjustments to volumes and dilutions. Please contact customer service at 313­961­1606 for assistance.
I.

Protocol for 1.0 mL plasma or serum
1. Combine 1.0 mL of plasma (adjusted with approximately 12 μL of acetic acid to pH 4) and 1.0
mL of ethyl acetate. Vortex thoroughly. Centrifuge at 2000 rpm for ten minutes at 22°C. Threephases should result:i. Upper organic phase – ethyl acetate phase (lipoproteins)ii. Interphase – proteinsiii. Lower phase – aqueous phase
2. Collect the upper organic phase (a) and set aside.
3. Discard the interphase. Transfer the lower phase with a glass pipette to a new tube, and repeat
the ethyl acetate extraction step 2 more times.
4. Evaporation of pooled organic phase: There should be approximately 3 mL of the ethyl
acetate phase (a). Dry the pooled organic phase in a Speedvac to get the extracted sediment(b).
5. Saponification (to cleave fatty acid from glycerol backbone): Dissolve the dried residues (b)in 2 mL of 20% KOH solution (for preparation see 11,12­DHET measurement in cells).
Vortex thoroughly and incubate for 1 h at 50°C. This will yield an aqueous solution (c).
6. Dilute 2 mL of the aqueous solution (c) with 3 mL of H2O. Adjust the pH using 20% formic
acid (132 μL) to pH~5.5. Add ethyl acetate (1 part aqueous solution (c) + 1 part ethyl acetate),
vortex thoroughly, and centrifuge at 2000 rpm for ten minutes at 22°C. Repeat the procedure
twice more using an equal volume of ethyl acetate per sample. Collect the upper phase
containing saponified lipids.
7. Dry the pooled ethyl acetate upper phase (d) and dry in a Speedvac, yielding the dried sample­
sediment (e). Store the sediment (e) at ­20°C. For ELISA assay, dissolve the sediment (e) in
20 μL of ethanol, then add 380 μL of 1X Sample Dilution Buffer, pH 7.4. (Please note thatthe 10X Sample Dilution Buffer that is supplied with the ELISA kit must be diluted 10­fold).

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Page 4 of 9

8.

When calculating the concentration, consider the dilution factor. In this case, 400 μL total
sample volume from 1.0 mL plasma (2.5­fold concentration) you must divide your calculated
result by 2.5.
Perform the ELISA for 11,12­DHET (according to the instructions of the manufacturer).

9.

II. Protocol for 1.8 mL plasma or serum (Following this procedure, the user will have approximately 70
uL of material left­over from step 8. This material can be stored at ­20 oC and be used for a second
measurement following a 5X dilution).
1.Combine 1.8 mL of plasma (adjusted with approximately 20 μL of acetic acid to pH 4) and 1.8
mL of ethyl acetate. Vortex thoroughly. Centrifuge at 2000 rpm for ten minutes at 22°C. Threephases should result:i. Upper organic phase – ethyl acetate phase (lipoproteins)ii. Interphase – proteinsiii. Lower phase – aqueous phase
2. Collect the upper organic phase (a) and set aside.
3. Discard the interphase. Transfer the lower phase with a glass pipette to a new tube, andrepeat the ethyl acetate extraction step 2 more times.
4. Evaporation of pooled organic phase: There should be approximately 5­6 mL of the ethyl
acetate phase (a). Dry the pooled organic phase in a Speedvac to get the extracted sediment(b).
5. Saponification (to cleave fatty acid from glycerol backbone): Dissolve the dried residues (b)in 2 mL of 20% KOH solution (for preparation see 11,12­DHET measurement in cells).
Vortex thoroughly and incubate for 1 h at 50°C. This will yield an aqueous solution (c).
6. Dilute 2 mL of the aqueous solution (c) with 3 mL of H2O. Adjust the pH using 20%
formic acid (132 μL) to pH~5.5. Add ethyl acetate (1 part aqueous solution (c) + 1 part
ethyl acetate), vortex thoroughly, and centrifuge at 2000 rpm for ten minutes at 22°C.
Repeat the procedure twice more using an equal volume of ethyl acetate per sample.
Collect the upper phase containing saponified lipids.
7. Dry the pooled ethyl acetate upper phase (d) and dry in a Speedvac, yielding the driedsample­sediment (e). Store the sediment (e) at ­20°C. For ELISA assay, dissolve thesediment (e) in 20 μL of ethanol, then add 130 μL of 1X Sample Dilution Buffer.
8. For the competitive 11,12­DHET ELISA, the above 150 μL sample needs to be further
diluted: Dilute 1:4 (e.g., 80 μL sample + 320 μL 1x Sample Dilution Buffer). Check the
final pH (should be pH 7.4). When calculating the concentration, consider the dilution
factor. In this case, 150 μL total sample volume from 1.8 mL plasma (12­foldconcentration) and then, 80 uL sample in 400 μL SDB (5­fold dilution). Since, the samples
are concentrated 2.4­fold; to get the actual concentration, you must divide by 2.4.
9. Perform the ELISA for 11,12­DHET (according to the instructions of the manufacturer).

11,12­DHET measurement in urine.
1.

2.
3.

Extraction using ethyl acetate is not necessary. It is recommended that the urine sample bediluted 4­fold with 1X sample dilution buffer and 100 uL of sample added directly to the
ELISA plate well.
However, extraction of urine with ethyl acetate can be performed if desired. See protocols
for extraction with ethyl acetate above.
When calculating the concentration, consider the dilution factor

Please contact us for urine or plasma extraction procedure or human study.

Assay Preparations

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Page 5 of 9

The solid 96­well plate and TMB solution are provided ready to use. The preparations of other assay reagents aredetailed below.
Wash Buffer: Mix the solution with a stir bar, applying low heat until a clear colorless solution is obtained. Dilutethe entire contents of the Wash Buffer Concentrate (25 mL) with 225 mL of deionized water to yield a final volumeof 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 11,12­DHET­HRP 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
11,12­DHET standard vial (2 μL, filled with inert gas) and add 1.998 mL of Sample Dilution Buffer to obtain 2 mLof 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 toserially 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 anddried samples, it is recommended to dissolve the dried­up samples with a minimal amount of ethanol of N, N­
dimethyl­formanmide (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.

Performing the Assay
Plate Setup: Each plate must contain a minimum of three blank wells (B L), three maximum binding wells (BO ), anda six point standard curve (S1­S 6). 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—S 6

=SamplesStandard Dilutions Table

2727 Second Ave. Suite 4113 – Detroit, MI 48201 – tel. 313.961.1606 – fax. 313.963.7130 – info@detroitrandd.com – www.detroitRandD.com

Page 6 of 9Standards
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 (B L) wells and 100 microliters ofSample 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 11,12­DHET­HRP 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.
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 B L wells).
Step 9: Incubate the plate at room temperature for 15­30 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 thesample concentrations. If your plate reader does not have this option, then a data reduction program can be used (4parameter of log­log 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 obtainthe 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 S 1 wells, divide theaverage by the maximum binding, then multiply by 100. Repeat this formula for the remaining standards.
Plot the %B/B O versus the concentration of 11,12­DHET from the standards using semi­log 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.

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Page 7 of 9

Typical Results
100
90
80

% B /B 0

70
60
50
40
30
20
10
0
1

10

100
1000
10000 100000 1000000
11, 12 DHET Conc. pg/m L

The data shown here is an example of typical results obtained using the
Detroit R & D 11,12­DHET ELISA kit. These results are only aguideline, and should not be used to determine values from your samples.
The user must run their own standard curve every time.
B L wells
B O wells= 0.066= 1.337

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.
1.179
1.126
0.981
0.495
0.108
0.019

%B/B O
88.2
84.2
73.4
37.1
8.1
1.4

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 propertime.
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 isreached.
The color is faint.
§ One or all of the incubation times were cut short. Redo the assay with the proper incubation times.

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Page 8 of 9§§

The TMB substrate was not warmed up to room temperature. Redo the assay making sure all reagentsare at room temperature.
The lab is too cold. Be sure the lab temperature is between 21­27°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 8­channel pipetman making sure that 8 channels are equal volume while loading.

References
1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

Kim et al. Two divisional US Patents: 6,440,682 and 6,534,282 issued on 8/27/2002 and 3/18/2003,
respectively.
Garson et al. Computational characterization of a series of eicosanoids. Lett. Drug Design and Discovery 2,
322, 2005.
Sinal et al. Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation. J. Biol.
Chem. 275, 40504, 2000.
Yu et al. Soluble epoxide hydrolase regulates hydrolysis of vascoactive epoxyeicosatrienoic acids. Circulation
Research 87, 992, 2000.
Makita et al. Cytochrome P450, the arachidonic acid cascade, and hypertension: new vistas for an old enzyme
system. FASEB J. 10, 1456, 1996, and references therein.
Spiecker et al. Risk of coronary artery disease associated with polymorphism of the cytochrome P450
epoxygenase CYP2J2. Circulation 110, 2132, 2004.
Wang et al. Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up­regulated inhuman tumors. Cancer Res. 65, 4707, 2005.
Wang et al. Cytochrome P­450 Epoxygenase Promotes Human Cancer Metastasis. Cancer Res. 67, 6665­6667,
July 15, 2007.
Zeldin et al. Cytochrome P­450 epoxygenases protect endothelial cells from apoptosis induced by tumor
necrosis factor­a via MAPK and PI3K/Akt signaling pathways. Am J. Physiol Heart Circ Physiol 293: H142­
H151, 2007.
Alkayed et al. Polymorphisms in the Human Soluble Epoxide Hydrolase Gene EPHX2 Linked to Neuronal
Survival after Ischemic Injury. J. Neurosci. 27(17): 464­4649, April 25, 2007.
Chen et al. Synergistic effect of cytochrome P450 epoxygenase CYP2J2*7 polymorphism with smoking on theonset of premature myocardial infraction. Atherosclerosis 195, 199­206, 2007.
D. Wang, T. Hirase, T. Nitto, M. Soma, K. Node. Eicosapentaenoic acid increases cytochrome P450 2J2 gene
expression and epoxyeicosatrienoic acid production via peroxisome proliferator­activated receptor g inendothelial cells. J. Cardiol. 54, 368­374, 2009.

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.

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Page 9 of 9

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
E­mail: 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