OxiSelect™ ORAC Activity Assay Kit

Catalog No. ABIN2345006

Product Details

Reactivity: Others

Detection Method: Fluorometric

Application: Activity Assay (AcA)

Brand: OxiSelect™

Sample Type: Plasma

Characteristics: OxiSelect™ ORAC Activity Assay is a fast and reliable kit for the direct measurement of ORAC antioxidant capacity from cell lysate, plasma, serum, tissue homogenates, and food extracts. Each kit provides sufficient reagents to perform up to 192 assays, including blanks, antioxidant standards and unknown samples. The assay is designed for use in single plate microplate readers as well as readers with high-throughput capabilities. Please read the complete kit insert prior to performing the assay.

Components:

1. 96-well Microtiter Plate : Two 96-well clear bottom black plates.
2. Fluorescein Probe (100X) : One 0.5 mL vial.
3. Free Radical Initiator : One 0.5 g bottle of powder.
4. Antioxidant Standard (Trolox™) : One 100 μL vial of a 5 mM solution.
5. Assay Diluent (4X) : One 50 mL bottle.

Material not included:

1. Sample extracts for testing
2. 1X PBS and Deionized water
3. 50 % Acetone
4. 37 °C incubator
5. Bottles, flasks, and conical or microtubes necessary for reagent preparation
6. Reagents and materials necessary for sample extraction and purification
7. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
8. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
9. Multichannel micropipette reservoir
10. Fluorescence microplate reader equipped with a 480 nm excitation filter and 520 nm emission filter

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Obtain results in less than 2 hours
• Suitable for use with plasma, cell fractions, tissue lysates, solid and aqueous nutrition samples
• Antioxidant standard included

Reagent Preparation:

• 1X Assay Diluent: Dilute the Assay Diluent 1:4 with deionized water. Mix to homogeneity. Use this for all sample and standard dilutions. Store the 1X Assay Diluent at 4 °C.
• 1X Fluorescein Probe: Dilute the Fluorescein Probe 1:100 with 1X Assay Diluent. Mix to homogeneity. Label this as 1X Fluorescein Solution. Use only enough Fluorescein Probe as necessary for immediate applications. Note: Do not store diluted Fluorescein Probe solutions.
• Free Radical Initiator Solution: Freshly prepare 80 mg/mL Free Radical Initiator Solution in 1X PBS. For example, weigh out 160 mg of Free Radical Initiator powder in a conical tube and reconstitute the powder with 2 mL of 1X PBS and mix to homogeneity. Free Radical Initiator Solution is not stable and should be used immediately.

Sample Preparation:

Note: Samples should be stored at -70 °C prior to performing the assay. Sample should be prepared at the discretion of the user. The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design.

• Deproteinated Fractions: Samples can be deproteinated and have their non-protein fractions assayed. Mix samples with 0.5 M perchloric acid (1:2, v/v), centrifuge at 10,000 x g for 10 minutes at 4 °C. Remove the supernatant for measuring the non-protein fraction in the assay.
• Cell Culture: Wash cells 3 times with cold PBS prior to lysis. Lyse cells with sonication or homogenation in cold PBS and centrifuge at 10,000 x g for 10 minutes at 4 °C. Aliquot and store the supernatant for use in the assay.
• Lipophilic Fractions: Dissolve lipophilic samples in 100 % acetone and then dilute in 50 % acetone. Incubate the mixture for 1 hour at room temperature with mixing. Further dilute samples as necessary prior to testing.
• Plasma or Serum: Collect blood with heparin and centrifuge at 4 °C for 10 minutes. Remove the plasma and aliquot samples for testing. Blood plasma or serum should be diluted 100-fold or more with Assay Diluent prior to performing the assay.
• Tissue Lysate: Sonicate or homogenize tissue sample on cold PBS and centrifuge at 10,000 x g for 10 minutes at 4 °C. Aliquot and store the supernatant for use in the assay.
• Urine: Test neat or diluted with Assay Diluent if appropriate.
• Nutrition Samples: Results may vary depending on sample source and purification. Dilution and preparation of these samples is at the discretion of the user, but use the following guidelines: o Solid or High Protein Samples: Weigh solid sample and then homogenize after adding deionized water (1:2, w/v). Centrifuge the homogenate at 10-12,000 x g for 10 minutes at 4 °C. Recover the supernatant which is the water-soluble fraction. Separately recover the insoluble fraction (pulp) and wash with deionized water. Combine this wash with the supernatant. The pooled supernatant can be diluted with Assay Diluent and used directly in the assay. The pulp is further extracted by adding pure acetone (1:4, w(solid pulp)/v) and mixing at room temperature 4 for 30-60 minutes. Centrifuge the extract/solid at 12,000 x g for 10 minutes at 4 °C. Recover the acetone extract and dilute with Assay Diluent as necessary prior to running the assay. The total ORAC value is calculated by combining the results from the water-soluble fraction and the acetone extract from the pulp fraction. o Aqueous Samples: Centrifuge the sample at 5-10,000 x g for 10 minutes at 4 °C to remove any particulates. Dilute the supernatant as necessary prior to running the assay. Certain liquids such as juice extracts may be tested without dilution.

Assay Procedure:

Note: Each Antioxidant Standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 25 μL of the diluted Antioxidant Standard or samples to the 96-well Microtiter Plate.
2. Add 150 μL of the 1X Fluorescein Solution to each well. Mix thoroughly. Incubate the plate for 30 minutes at 37 °C.
3. Add 25 μL of the Free Radical Initiator Solution into each well using either a multichannel pipette or a plate reader liquid handling system.
4. Mix the reaction mixture thoroughly by pipetting to ensure homogeneity.
5. Immediately begin reading sample and standard wells with a fluorescent microplate reader at 37 °C with an excitation wavelength of 480nm and an emission wavelength of 520nm. Read the wells in increments between 1 and 5 minutes for a total of 60 minutes. Save values for Calculation of Results below. Note: The final assay values of blank control should be less than 10 % of the initial values in order for the assay to be completed.

Calculation of Results:

Note: A spreadsheet application or plate reader software can be used to perform the calculations.

1. Calculate the area under the curve (AUC) for each sample and standard using the final assay values and the linear regression formula below. The AUC can be calculated from the equation below: AUC = 1 + RFU1/RFU0 + RFU2/RFU0 + RFU3/RFU0 +......+ RFU59/RFU0 + RFU60/RFU0 RFU0 = relative fluorescence value of time point zero. RFUx = relative fluorescence value of time points (eg. RFU5 is relative fluorescence value at minute five) AUC (Blank) AUC (Antioxidant) 1.2 1.2 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 0 5 10 15 20 25 30 35 40 45 50 55 60 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) Time (min)
2. Calculate the Net AUC by subtracting the Blank AUC from the AUC of each sample and standard. Net AUC = AUC (Antioxidant) - AUC (blank) 7 RFU RFU RFU Net AUC Net AUC 1.20 1.00 0.80 0.60 0.40 0.20 0.00 5 10 15 20 25 30 35 40 45 50 55 60 Time (min)
3. Graph the Net AUC on the y-axis against the Trolox™ Antioxidant Standard concentration on the x-axis (see Figure 1)
4. Calculate the μMole Trolox™ Equivalents (TE) of unknown sample by comparing the standard curve. Results (ORAC value) may be expressed as TE per L or g of sample. Calculation Example: 25 μL of 10-fold diluted sample is assayed along with 25 μL of each Trolox™ antioxidant standard including blank as described in Assay Protocol. The average AUC is 4.3 for blank and 9.1 for sample. Net AUC = AUC (Antioxidant) - AUC (blank) = 9.1 - 4.3 = 4.8 Based on the Trolox™ antioxidant standard curve, the equivalent Trolox concentration is 20 μM, therefore: ORAC value (Sample) = 20 μM x 10 (dilution factor) = 200 μM TE = 200 μMole TE/L

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt store the Fluorescent Probe and Antioxidant Standard (Trolox™) frozen at -20°C. Aliquot as necessary to avoid multiple freeze/thaws. Store all remaining components at 4°C. 3

References

Konishi, Baumgarten, Ishida, Saitoh, Anker, Springer: "Protein levels in Keap1-Nrf2 system in human failing heart." in: International journal of cardiology, Vol. 225, pp. 62-64, (2016) (PubMed).

Alleva, Manzella, Gaetani, Ciarapica, Bracci, Caboni, Pasini, Monaco, Amati, Borghi, Tomasetti: "Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response." in: Molecular nutrition & food research, Vol. 60, Issue 10, pp. 2243-2255, (2016) (PubMed).

Xiao, Miwa et al.: "The Lipophilic Vitamin C Derivative, 6-O-Palmitoylascorbate Protects Human Keratinocytes and 3D-Human Skin Equivalents Against X-Ray-Induced Oxidative Stress and Apoptosis More Markedly Than ..." in: Journal of cellular biochemistry, Vol. 118, Issue 2, pp. 318-329, (2016) (PubMed).

Xiao, Miwa et al.: "Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging ..." in: Human cell, Vol. 30, Issue 2, pp. 72-87, (2016) (PubMed).

Pate, Rogers, Reed, van der Munnik, Vance, Moss: "Anthoxanthin Polyphenols Attenuate Aβ Oligomer-induced Neuronal Responses Associated with Alzheimer's Disease." in: CNS neuroscience & therapeutics, Vol. 23, Issue 2, pp. 135-144, (2016) (PubMed).

Gao, Park, Cha, Bae, Park, Park, Kim: "Oxidative stress increases the risk of pancreatic β cell damage in chronic renal hypertensive rats." in: Physiological reports, Vol. 4, Issue 16, (2016) (PubMed).

Hutchison, Flieller, Dillon, Leverett: "Black Currant Nectar Reduces Muscle Damage and Inflammation Following a Bout of High-Intensity Eccentric Contractions." in: Journal of dietary supplements, Vol. 13, Issue 1, pp. 1-15, (2015) (PubMed).

Wada, Kubota, Sawa, Umekita, Hatano, Ohba, Hayashi, Igarashi, Nomoto: "Novel autophagy inducers lentztrehaloses A, B and C." in: The Journal of antibiotics, Vol. 68, Issue 8, pp. 521-9, (2015) (PubMed).

Macchi, Wang, Moore, Katz, Saperstein, Walk, Simpson, Genge, Bertorini, Fernandes, Swenson, Elman, Dimachkie, Herbelin, Miller, Lu, Wilkins, Swerdlow, Statland, Barohn: "A multi-center screening trial of rasagiline in patients with amyotrophic lateral sclerosis: Possible mitochondrial biomarker target engagement." in: Amyotrophic lateral sclerosis & frontotemporal degeneration, Vol. 16, Issue 5-6, pp. 345-52, (2015) (PubMed).

Orena, Owen, Jin, Fabian, Gillitt, Zeisel: "Extracts of Fruits and Vegetables Activate the Antioxidant Response Element in IMR-32 Cells." in: The Journal of nutrition, Vol. 145, Issue 9, pp. 2006-11, (2015) (PubMed).

Nishikawa, Furukawa, Shiga, Muroi, Ishii, Hongo, Takahashi, Sugawara, Koshino, Ohnishi: "Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra." in: PLoS ONE, Vol. 10, Issue 8, pp. e0135701, (2015) (PubMed).

Okutsu, Yoshizaki, Ikeda, Kusano, Hashimoto, Takamine: "Antioxidants in heat-processed koji and the production mechanisms." in: Food chemistry, Vol. 187, pp. 364-9, (2015) (PubMed).

Yang, Li, Chan, Tsai, Wu, Nguyen, Hsu, Li, Brown, Egli, Sparrow, Tsang: "Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines." in: Human molecular genetics, Vol. 23, Issue 13, pp. 3445-55, (2014) (PubMed).

Han, Lin, Lin, Lee, Huang, Lin, Hou: "Effects of yam tuber protein, dioscorin, on attenuating oxidative status and learning dysfunction in d-galactose-induced BALB/c mice." in: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, Vol. 65, pp. 356-63, (2014) (PubMed).

Yoo, Kang, Yun, Kim, Nho: "Crepidiastrum denticulatum extract protects the liver against chronic alcohol-induced damage and fat accumulation in rats." in: Journal of medicinal food, Vol. 17, Issue 4, pp. 432-8, (2014) (PubMed).

Krautbauer, Eisinger, Hader, Neumeier, Buechler: "Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis." in: Molecular and cellular biochemistry, Vol. 393, Issue 1-2, pp. 69-76, (2014) (PubMed).

Han, Lin, Lee, Liang, Hou: "Asn-Trp dipeptides improve the oxidative stress and learning dysfunctions in D-galactose-induced BALB/c mice." in: Food & function, Vol. 5, Issue 9, pp. 2228-36, (2014) (PubMed).

Krautbauer, Eisinger, Neumeier, Hader, Buettner, Schmid, Aslanidis, Buechler: "Free fatty acids, lipopolysaccharide and IL-1α induce adipocyte manganese superoxide dismutase which is increased in visceral adipose tissues of obese rodents." in: PLoS ONE, Vol. 9, Issue 1, pp. e86866, (2014) (PubMed).

Han, Lin, Lin, Hou: "Antioxidant and antiglycation activities of the synthesised dipeptide, Asn-Trp, derived from computer-aided simulation of yam dioscorin hydrolysis and its analogue, Gln-Trp." in: Food chemistry, Vol. 147, pp. 195-202, (2013) (PubMed).

Ungvari, Csiszar, Sosnowska, Philipp, Campbell, McQuary, Chow, Coelho, Didier, Gelino, Holmbeck, Kim, Levy, Sonntag, Whitby, Austad, Ridgway: "Testing predictions of the oxidative stress hypothesis of aging using a novel invertebrate model of longevity: the giant clam (Tridacna derasa)." in: The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 68, Issue 4, pp. 359-67, (2013) (PubMed).

Target Details

Background: Oxidative stress is a physiological condition where there is an imbalance between concentrations of reactive oxygen species (ROS) and antioxidants. However, excessive ROS accumulation will lead to cellular injury, such as damage to DNA, proteins, and lipid membranes. The cellular damage caused by ROS has been implicated in the development of many disease states, such as cancer, diabetes, cardiovascular disease, atherosclerosis, and neurodegenerative diseases. Under normal physiological conditions, cellular ROS generation is counterbalanced by the action of cellular antioxidant enzymes and other redox molecules. Because of their potential harmful effects, excessive ROS must be promptly eliminated from the cells by this variety of antioxidant defense mechanisms. Antioxidants include both hydrophilic and lipophilic molecules for metabolizing ROS. Although the products of ROS-induced oxidative stress are extensively used to monitor the effects of oxidative stress, it is also important to evaluate the antioxidant capacity of biological fluids, cells, and extracts. The Oxygen Radical Antioxidant Capacity (ORAC) Assay is a classic tool for measuring the antioxidant capacity of biomolecules from a variety of samples. The ORAC Activity Assay is based on the oxidation of a fluorescent probe by peroxyl radicals by way of a hydrogen atom transfer (HAT) process. Peroxyl radicals are produced by a free radical initiator, which quenches the fluorescent probe over time. Antioxidants present in the assay work to block the peroxyl radical oxidation of the fluorescent probe until the antioxidant activity in the sample is depleted. The remaining peroxyl radicals destroy the fluorescence of the fluorescent probe. This assay continues until completion, which means both the antioxidant's inhibition time and inhibition percentage of free radical damage is a single value. The sample antioxidant capacity correlates to the fluorescence decay curve, which is usually represented as the area under the curve (AUC). The AUC is used to quantify the total peroxyl radical antioxidant activity in a sample and is compared to an antioxidant standard curve of the water soluble vitamin E analog Trolox™ (see Assay Principle below).