Human 12-HETE12-Hydroxyeicosatetraenoic Acid ELISA Kit

12-Hydroxyeicosatetraenoic Acid (12-HETE) ELISA Kit is an ELISA Kit for the in vitro quantitative measurement of 12-Hydroxyeicosatetraenoic Acid (12-HETE) concentrations in serum, plasma and other biological fluids.Human 12-hydroxyeicosatetraenoic acid (12-HETE) ELISA Kit has high sensitivity and excellent specificity for detection of Human 12-HETE. No significant cross-reactivity or interference between Human 12-HETE and analogues was observed. 12-Hydroxyeicosatetraenoic acid (12-HETE) is an eicosanoid. It is a metabolite of arachidonic acid produced by the action of the enzyme arachidonate 12-lipoxygenase. 12-HETE is a highly selective ligand used to label mu opioid receptors.

12-HETE can activate Cdc42, Rac1 and PI-3K, which then participate as upstream signalling molecules for PAK1 and JNK1 activation. 12-Hydroxyeicosatetraenoic acid (12-HETE) is a chemotactic stimulus for epidermal cells. 12-hydroxyeicosatetraenoic acid (12-HETE), the main eicosanoid in skin, is assumed to have both pathophysiologic effects in inflammatory skin diseases such as psoriasis and atopic eczema and a physiologic role in the biology of cutaneous reparative processes. The modulation of epidermal 12-HETE receptors by UV-B may partly explain the therapeutic effects of UV-B, but possibly also contribute to photodamage to skin.

This Human 12-hydroxyeicosatetraenoic acid (12-HETE) ELISA Kit employs a two-site sandwich ELISA to quantitate 12-HETE in samples. An antibody specific for 12-HETE has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any12-HETE present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for 12-HETE is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of 12-HETE bound in the initial step Bioscience. The color development is stopped and the intensity of the color is measured.

Target 12-Hydroxyeicosatetraenoic Acid (12-HETE)
Reactivity General (All species)
Tested Applications ELISA
Recommended dilutions Optimal dilutions/concentrations should be determined by the end user.
Storage Shipped at 4 °C. Upon receipt, store the kit according to the storage instruction in the kit’s manual.
Validity The validity for this kit is 6 months.
Stability The stability of the kit is determined by the rate of activity loss. The loss rate is less than 5% within the expiration date under appropriate storage conditions. To minimize performance fluctuations, operation procedures and lab conditions should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same user throughout.
Test Range 0.78 ng/ml – 50 ng/ml
Sensitivity 0.47 ng/ml
Standard Form Lyophilized
Detection Method Colorimetric
Assay Type Competitive
Assay Data Quantitative
Sample Type Serum, plasma and other biological fluids.
Kit Components The kit components listed are for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.

  • Pre-coated 96-Well Microplate
  • Standard
  • Standard Diluent Buffer
  • Wash Buffer
  • Detection Reagent A
  • Detection Reagent B
  • Diluent A
  • Diluent B
  • TMB Substrate
  • Stop Solution
  • Plate Sealer
Material Required But Not Provided
  • 37°C incubator
  • Multi and single channel pipettes and sterile pipette tips
  • Squirt bottle or automated microplate washer
  • 1.5 ml tubes
  • Distilled water
  • Absorbent filter papers
  • 100 ml and 1 liter graduated cylinders
  • Microplate reader (wavelength: 450 nm)
  • ELISA Shaker
Sample Collection/Preparation
  • Serum: Samples should be collected into a serum separator tube. Coagulate the serum by leaving the tube undisturbed in a vertical position overnight at 4°C or at room temperature for up to 60 minutes. Centrifuge at approximately 1000 × g for 20 min. Analyze the serum immediately or aliquot and store at -20°C or -80°C.
  • Plasma: Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for 15 minutes at 1000 × g within 30 minutes of collection. Assay immediately or aliquot and store at -20°C or -80°C. Avoid hemolysis and high cholesterol samples.
  • Tissue homogenates: The preparation of tissue homogenates will vary depending upon tissue type – this is just an example. Rinse tissues with ice-cold PBS to remove the excess of blood. Weigh before homogenization. Finely mince tissues and homogenize with a tissue homogenizer on ice in PBS and sonicate the cell suspension. Centrifuge the homogenates at 5000 × g for 5 min and collect the supernatant. Assay immediately or aliquot and store at -20°C.
Reagent Preparation This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.

  • 1) Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the Standard Diluent buffer to carry out serial dilutions of the standard solution, as instructed in the Protocol.
  • 2) Wash Buffer: Dilute the concentrated Wash Buffer with distilled water, as instructed in the Protocol.
  • 3) Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, at 1:100.
Assay Procedure This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.

  • 1) Set standard, test samples and control wells.
  • 2) Aliquot 50 µl of diluted standard into the standard wells.
  • 3) Aliquot 50 µl of Standard Diluent buffer into the control (zero) well.
  • 4) Aliquot 50 µl of diluted samples into the sample wells.
  • 5) Immediately aliquot 50 µl of Detection Reagent A to each well. Incubate for 45 mins at 37 °C.
  • 6) Wash 3 times.
  • 7) Aliquot 100 µl of Detection Reagent B to each well. Incubate for 30 mins at 37 °C.
  • 8) Wash 5 times.
  • 9) Aliquot 90 µl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C.
  • 10) Aliquot 50 µl of Stop Solution.
  • 11) Measure the OD at 450 nm.
Assay Precision Intra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of Interleukin 10 (IL10) were were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of Interleukin 10 (IL10) were tested on 3 different plates, 8 replicates in each plate.

CV (%) = (Standard Deviation / mean) × 100

Intra-Assay: CV<10%

Inter-Assay: CV<10%

Availability Shipped within 5-12 working days.
Note This product is for research use only. 

The range and sensitivity is subject to change. Please contact us for the latest product information. For accurate results, sample concentrations must be diluted to mid-range of the kit. If you require a specific range, please contact us in advance or write your request in your order comments. 

Please note that our ELISA and CLIA kits are optimised for detection of native samples, rather than recombinant proteins. We are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native protein.


This assay employs the competitive inhibition enzyme immunoassay technique.

The micro-plate provided in this kit has been pre-coated with 12-Hydroxyeicosatetraenoic Acid (12-HETE) protein. Standards or samples are then added to the appropriate micro-plate wells with a biotin-conjugated antibody specific to 12-Hydroxyeicosatetraenoic Acid (12-HETE). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm±10nm. The concentration of 12-Hydroxyeicosatetraenoic Acid (12-HETE) in the samples is then determined by comparing the OD of the samples to the standard curve.

Kit Components:
1) Pre-coated Microplate [12 strips x 8 wells]
2) 100x Biotinylated Conjugate [120ul]
3) Biotinylated Conjugate Diluent [10ml]
4) 100x Streptavidin-HRP [120ul]
5) HRP Diluent [12ml]
6) TMB Substrate Solution [9ml]
7) 25x Wash Buffer [20ml]
8) Stop reagent [6ml]
9) Lyophilized Standard [2]
10) Standard Diluent Buffer [20ml]
11) Plate Sealers [2]
12) User Maual [1]

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