Oxidative RNA Damage ELISA Kit

Details for Product No. ABIN2344974, Supplier: Log in to see
Antigen
Reactivity
Others
Method Type
Competition ELISA
Application
ELISA
Options
Supplier
Log in to see
Supplier Product No.
Log in to see
Purpose The Oxidative RNA Damage ELISA kit is a competitive ELISA for the quantitative measurement of 8- OHG. The unknown 8-OHG samples or 8-OHG standards are first added to an 8-OHG/BSA conjugate preabsorbed EIA plate. After a brief incubation, an anti-8-OHG monoclonal antibody is added, followed by an HRP conjugated secondary antibody. The 8-OHG content in unknown samples is determined by comparison with predetermined 8-OHG standard curve.
Brand OxiSelect™
Sample Type Urine, Plasma, Cell Samples, Cerebrospinal Fluid, Serum, Tissue Samples
Analytical Method Quantitative
Detection Method Colorimetric
Characteristics The OxiSelect™ Oxidative RNA Damage ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of 8-OHG in urine, serum, cerebrospinal fluid or other cell or tissue RNA samples. The quantity of 8-OHG in unknown sample is determined by comparing its absorbance with that of a known 8-OHG standard curve. The kit has an 8-OHG detection sensitivity range of 300 pg/mL to 40 ng/mL. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.
Components
  1. 96-well Protein Binding Plate : One strip well 96-well plate.
  2. Anti-8-OHG Antibody : One 15 μL vial of anti-8-OHG.
  3. Secondary Antibody, HRP Conjugate (1000X) : One 20 μL vial.
  4. Assay Diluent : One 50 mL bottle.
  5. 10X Wash Buffer : One 100 mL bottle.
  6. Substrate Solution : One 12 mL amber bottle.
  7. Stop Solution (Part. No. 310808): One 12 mL bottle.
  8. 8-OHG Standard : One 100 μL vial of 2 μg/mL 8-OHG in 1X PBS, 0.1% BSA.

Box 2 (shipped on blue ice packs)

Material not included
  1. 8-OHG samples such as serum, urine, cerebrospinal fluid, cell or tissue RNA
  2. RNA Extraction Kit
  3. Sodium Acetate, pH 5.2
  4. Tris Buffer, pH 7.5
  5. Nuclease P1, Alkaline Phosphatase
  6. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  7. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  8. Multichannel micropipette reservoir
  9. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
Background Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nuclei acids, proteins and lipids. RNA and DNA are probably the most biologically significant targets of oxidative attack. Recently, oxidative damage to RNA molecules has been described in several neurological diseases including Alzheimer's disease, Parkinson's disease, Down syndrome, dementia with Lewy bodies, prion disease, subacute sclerosing panencephalitis, and xeroderma pigmentosum. Among numerous types of RNA oxidative damage, the formation of 8-hydroxyguanosine (8-OHG) is a ubiquitous marker of oxidative stress.
Comment

  • ELISA detects 300 pg/mL to 40 ng/mL of 8-OHG
  • Suitable for use with RNA from urine, serum, cerebrospinal fluid, cells or tissues
  • 8-OHG standard included for absolute quantitation

Plate Uncoated
Reagent Preparation
  • 8-OHG Coated Plate: Dilute the proper amount of 8-OHG Conjugate (1 mg/mL) to 1 μg/mL in 1X PBS. Add 100 μL of the 1 μg/mL 8-OHG Conjugate to each well and incubate overnight at 4 °C. Remove the 8-OHG coating solution and wash once with dH2O. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1 hr at room temperature. Transfer the plate to 4 °C and remove the Assay Diluent immediately before use. Note: 8-OHG coated plate is not stable. We recommend using it within 24 hrs after coating.
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
  • Anti-8-OHG Antibody and Secondary Antibody: Immediately before use dilute the Anti-8-OHG Antibody 1:500 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions. Preparation of Standard Curve Prepare a dilution series of 8-OHG standards in the concentration range of 0 ng/mL - 40 ng/mL by diluting the 8-OHG Standard in Assay Diluent (Table 1). Assay Diluent Standard Tubes 8-OHG Standard (μL) (μL) 8-OHG (ng/mL) 1 20 980 40 2 500 of Tube #1 500 20 3 500 of Tube #2 500 10 4 500 of Tube #3 500 5 5 500 of Tube #4 500 2.5 6 500 of Tube #5 500 1.25 7 500 of Tube #6 500 0.625 8 500 of Tube #7 500 0.313 9 500 of Tube #8 500 0.156 10 0 500 0 Table 1. Preparation of 8-OHG Standards
Sample Preparation

I. Urine, Serum, Plasma, or Cerebrospinal Fluid Samples (Quantitation of 8-OHG and 8-OHdG) Clear urine, serum or cerebrospinal fluid samples can be diluted in Assay Diluent and used directly in the assay. Samples containing precipitates should be centrifuged at 3000 g for 10 minutes, or filtered through 0.45 μm filter, prior to use in the assay. Note: For mouse or rat serum or plasma samples it is highly recommended to filter the sample with a 10 kDa spin filter prior to testing. 4 II. Cell or Tissue RNA Samples (Quantitation of 8-OHG):

  1. Extract RNA from cell or tissue samples by a desired method or commercial RNA Extraction kit.
  2. Dissolve extracted RNA in water at 1-5 mg/mL.
  3. Digest RNA sample to nucleosides by incubating the sample with 5-20 units of nuclease P1 for 2 hrs at 37 °C in 20 mM Sodium Acetate, pH 5.2, and following with treatment of 5-10 units of alkaline phosphatase for 1 hr at 37 0C in 100 mM Tris, pH 7.5.
  4. The reaction mixture is centrifuged for 5 minutes at 6000 g and the supernatant is used for the 8- OHG assay.

Assay Procedure
  1. Prepare and mix all reagents thoroughly before use. Each 8-OHG sample including unknown and standard should be assayed in duplicate. High content 8-OHG samples should be diluted with Assay Diluent.
  2. Add 50 μL of unknown sample or 8-OHG standard to the wells of the 8-OHG Conjugate coated plate. Incubate at room temperature for 10 minutes on an orbital shaker.
  3. Add 50 μL of the diluted anti-8-OHG antibody to each well, incubate at room temperature for 1 hour on an orbital shaker.
  4. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
  5. Add 100 μL of the diluted Secondary Antibody-Enzyme Conjugate to all wells.
  6. Incubate at room temperature for 1 hour on an orbital shaker.
  7. Wash microwell strips 3 times according to step 4 above. Proceed immediately to the next step.
  8. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
  9. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
  10. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 5
Restrictions For Research Use only
Handling Advice Avoid multiple freeze/thaw cycles.
Storage -20 °C/-80 °C
Storage Comment Upon receipt, aliquot and store the 8-OHG Standard at -20°C and the 8-OHG Conjugate at -80°C to avoid multiple freeze/thaw cycles. Store all other components at 4°C. 3
Supplier Images
ELISA image for Oxidative RNA Damage ELISA Kit (ABIN2344974) 8-OHG ELISA Standard Curve
Product cited in: Tsai, Liao, Chou, Contreras: "Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation." in: Applied and environmental microbiology, Vol. 81, Issue 5, pp. 1754-64, 2015 (PubMed).

Belenky, Ye, Porter, Cohen, Lobritz, Ferrante, Jain, Korry, Schwarz, Walker, Collins: "Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage." in: Cell reports, Vol. 13, Issue 5, pp. 968-80, 2015 (PubMed).

Giannakopoulos, Mirarabshahi, Qi, Weatherall, Qi, Tanaka, Millar, Vonthethoff, Gatto, Spielman, Krilis: "Deletion of the antiphospholipid syndrome autoantigen ?2 -glycoprotein I potentiates the lupus autoimmune phenotype in a Toll-like receptor 7-mediated murine model." in: Arthritis & rheumatology (Hoboken, N.J.), Vol. 66, Issue 8, pp. 2270-80, 2014 (PubMed).

Kannan, Dai, Navath, Balakrishnan, Jyoti, Janisse, Romero, Kannan: "Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model." in: Science translational medicine, Vol. 4, Issue 130, pp. 130ra46, 2012 (PubMed).

Bazin, Langlade, Vincourt, Arribat, Balzergue, El-Maarouf-Bouteau, Bailly: "Targeted mRNA oxidation regulates sunflower seed dormancy alleviation during dry after-ripening." in: The Plant cell, Vol. 23, Issue 6, pp. 2196-208, 2011 (PubMed).

Did you look for something else?