14,15­DHET Human Urine Test Kit
For measuring free and glucuronidated 14,15­DHET
Catalog Number: DH3, DH13, DH23, DH103
V. 10232012

Introduction
This competitive ELISA kit is for determination of free and glucuronidated 14,15‐DHET levels in urine. The
14,15‐DHET is a representative metabolite of soluble epoxide hydrolase‐mediated metabolism of EETs,
which are generated by arachidonic acid epoxygenase activity of cytochromes P450. 14,15‐DHET level
exhibited strong positive correlation with hypertension in rat and human and brain injury and stroke inrodents. Human urine and blood 14,15‐DHET levels were measured using the 14,15‐DHET ELISA kit.
High levels of the glucuronidated form of 14,15‐DHET have been found in human urine but not in urine
collected from rodents. This kit can be used for the determination of free and glucuronidated 14,15‐DHET inhuman urine following proper isolation and purification as provided in the following pages
This competitive ELISA kit, based on competition between the14,15­DHET epitope and the 14,15­DHET­HRP
conjugate for a limited number of binding sites available from the anti­14,15­DHET antibody, which is coated to thewells of the 96 well ELISA plate. The conjugate concentration is held as a constant in each well, while theconcentration of the 14,15­DHET is variable, based on the concentration of the sample or standard. Thus theamount of the 14,15­DHET conjugate which is able to bind to each of the wells is inversely proportional to theconcentration of 14,15­DHET in the standard or sample. The amount of the conjugate which is bound to each wellis then determined by the amount of color obtained, when TMB is added. The TMB reacts with the HRP available
in the well. With the addition of sulfuric acid, the blue colored product is converted into a yellow colored product,
which can be read on a plate 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 and should not be re­frozen and thawed more than necessary.

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.

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Page 2 of 9Materials Provided
Part
NumberItem

Description

Quantity

1

14,15­DHET ELISA Plate

Solid 96­well plate coated with anti­14,15­DHET antibody
in each well

1

14,15­DHET Standard(2 μL)
14,15­DHET­HRP
Conjugates(12 μL)
Sample Dilution Buffer(25 mL)HRP Buffer(15 mL)
Wash Buffer Solution(25 mL)TMB Substrate(24 mL)
Beta­Glucuronidase
enzyme

2
3
4
5
6
7
8

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

8 mg solid

1

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
2N Sulfuric Acid

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
Materials
Materials
Dissolve 8 mg of b‐glucuronidase (provided) in 8 mL of 1 M citric acid, adjust to pH 5.5. (400 U/mL).
Protocols
I. Measurement of A) free and B)glucuronidated 14,15‐DHET in sample by extraction with ethyl
acetate.
A.

Measurement of free 14,15‐DHET: Extract 4 mL of urine with ethyl acetate using theExtraction Protocol described below.

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Page 3 of 9

B. Measurement of free and 14,15‐DHET glucuronide
This method is for determining the level of glucuronidated 14,15‐DHET in urine after digestion of themolecule with glucuronidase. Collect the first sample as soon as the b‐glucuronidase is added to areaction mixture (0 hour digestion) and then a second sample at a time when digestion of the
glucuronic acid moiety of the molecule is completed. Subtract the level of the molecule in the firstsample at 0 hour from the levels in the second sample after complete digestion (usually 3 hrs) to obtainthe level of glucuronidated molecule.
b‐Glucuronidase digestion
1. To 4 mL of urine add 1 mL of the b‐glucuronidase solution, pH 5.5, to each tube (pH < 6.0).
2. Immediately transfer 2 mL of urine to a clean tube and flash‐freeze. This is the zero time point.
3. Incubate the remaining 2 mL at 37 oC for 3 hours. This is the 3‐hour time point.
4. Extract
5.
Extraction protocola) Combine an equal amount of urine sample from steps 2 and 3 above (adjusted with approximately
20 μL
of acetic acid to pH 4) and ethyl acetate. Vortex thoroughly. Centrifuge at 2000 rpm forten minutes at 22°C. Three phases should result:b) Upper organic phase – ethyl acetate phase (lipoproteins)c) Interphase – proteinsd) Lower phase – aqueous phasee) Collect the upper organic phase (a) and set aside.
f) Discard the interphase. Transfer the lower phase with a glass pipette to a new tube, and repeat theethyl acetate extraction step 2 more times.
g) 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).
h) Saponification (to cleave fatty acid from glycerol backbone): Dissolve the dried residues (b) in 2
mL of 20% KOH solution (for preparation see 14,15­DHET measurement in cells). Vortex
thoroughly and incubate for 1 h at 50°C. This will yield an aqueous solution (c).
i) 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 moreusing an equal volume of ethyl acetate per sample. Collect the upper phase containing saponified
lipids.
j) 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 μLof DMF or ethanol, then add 130 μL of 1X Sample Dilution Buffer.
k) For the competitive 14,15­DHET 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 final concentration, consider all dilution factors.
l) Perform the ELISA for 14,15­DHET according to the instructions of the manufacturer

II. Measurement of A) Free and B) Glucuronidated 14,15‐DHET without extraction
A. Measurement of free 14,15‐DHET: Dilute 1 mL urine 4‐fold with 1X Sample Dilution Buffer andapply to ELISA plate (100 µL/well). A 4X dilution is recommended although other dilution factors
may be tried, too.
B. Measurement of glucuronidated 14,15‐DHET
This method is for determining the level of glucuronidated 14,15‐DHET in urine after digestion of themolecule with glucuronidase. Collect the first sample as soon as the b‐glucuronidase is added to areaction mixture (0 hour digestion) and then a second sample at a time when digestion of the glucuronic

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Page 4 of 9acid moiety of the molecule is completed. Subtract the level of the molecule in the first sample at 0 hourfrom the levels in the second sample after complete digestion (usually 3 hrs) to obtain the level of
glucuronidated molecule.
b‐Glucuronidase digestion
6. Dilute 1 mL of urine 4‐fold with 1X Sample Dilution Buffer
7. To 4 mL of urine add 1 mL of the b‐glucuronidase solution, pH 5.5, to each tube (pH < 6.0).
8. Immediately transfer 2 mL of urine to a clean tube and flash‐freeze. This is the zero time point.
9. Incubate the remaining 2 mL at 37 oC for 3 hours. This is the 3‐hour time point.
10. Follow the instructions for the ELISA kit (see below).
11.To calculate the amount of glucuronidated 14,15‐DHET, subtract the 0 time value from the 3 hr value.
.

Assay Preparations
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 14,15­DHET­HRP 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
14,15­DHET 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:

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Page 5 of 9Standard Dilutions Table

Standards
No. 6
No. 5
No. 4
No. 3
No. 2
No. 1

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

Final Concentration (pg/mL)
1,000,000
100,000
10,000
1,000
100
10

=BO

=S1—S 6

Add Sample Dilution Buffer (mL)
1.998
0.9
0.9
0.9
0.9
0.9

=SamplesSerial 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 14,15­DHET­HRP conjugate in the BO wells, the standard wells,
and the sample wells. Do NOT add HRP conjugate into the B L 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.

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Page 6 of 9

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 14,15­DHET 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.

Typical Results

The data shown here is an example of typical results obtained using the
Detroit R & D 14,15­DHET 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= 0.059= 2.525

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Page 7 of 9Standard
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.
2.349
2.099
1.691
0.916
0.372
0.148

%B/B O
93.0
83.1
67.0
36.3
14.7
5.8

Specificity of anti-14,15-DHET IgG
The specificity of the 14,15­DHET ELISA was investigated using authentic 14,15­DHET and a panel ofeiconsanoids.
14,15­DHET
8,9­DHET
11,12­DHET
14,15­EET
15(s) HETE
8,9­EET5(s)15(s)DiHETE
11,12­EET
Arachidonic Acid
5,6­DHET
5,6­EET
Thromboxane B2
PGE2
PGF2a
6­keto­PGF1a

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% cross­reactivity.

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.

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Page 8 of 9

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 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. Spiecker et al. Risk of coronary artery disease associated with polymorphism of the cytochrome p450 epoxygenase CYP2J2.
Circulation 110, 2132, 2004.
2. Kim et.al. Computational characterization of a series of eicosanoids. Lett. Drug Design & Discovery 2: 322‐325. 2005
3. Wang et al. Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up‐regulated in human tumors. Cancer
Res. 65, 4707, 2005.
4. Zhang et.al. Effects of atorvastatin on expression of cytochrome P450 epoxygenase 2C11 in spontaneously hypertensive rats.
5. Alkayed, et al. Polymorphisms in the human soluble epoxide hydrolase gene EPHX2 linked to neuronal survival after ischemic injury.
The Journal of Neuroscience, 27(17): 464‐4649, April 25, 2007.
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7. Zeldin, et al. Cytochrome P‐450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor‐a via MAPK
and PI3K/Akt signaling pathways. Am J. Physiol Heart Circ Physiol 293: H142‐H151, 2007.
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10. Chen, Wang et al. Selective inhibitors of CYP2J2 related to terfenadine exhibit activity strongly against human cancers in vitro and invivo. J. Pharmacol. Exp. Ther. 329: 908‐918. 2009.
11. Alkayed et al. Role of soluble epoxide hydrolase in the sex‐specific vascular response to cerebral ischemia. J. Cerebral Blood Flow &Metabolism 29, 1475‐1481. 2009.
12. Eldrup et al. Structure‐based optimization of arylamides as inhibitors of soluble epoxide hydrolase. J. Med. Chem. 52: 5880‐5895,
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rats. Am. J. Respir. Cell Mol. Biol. 43: 740‐749, 2010.
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rats by enhancing atrial natriuretic peptide. J. Pharmaol. Exp. Ther. 334: 784‐794, 2010.
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Physiol. 298: H679‐H687, 2010.
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function in two‐kidney, one‐clip Goldblatt hypertensive rats. J Hypertens 28, 582‐593, 2010.
19. Wang et al. The epoxyeicosatrienoic acid‐stimulated phosphorylation of EGF‐R involves the activation of metalloproteinases and therelease of HB‐EGF in cancer cells. Acta Pharmacol Sin. 31, 211‐218, 2010.
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Diabetes. 118, 346‐352, 2010.

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

Page 9 of 9

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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
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