BPDE DNA Adduct ELISA Kit

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Antigen
Reactivity
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Method Type
DNA-Binding ELISA
Application
ELISA
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Purpose BPDE-DNA standards or unknown DNA samples are adsorbed onto a 96-well DNA high-binding plate. The BPDE-DNA adducts present in the sample or standard are probed with an Anti-BPDE-I Antibody, followed by an HRP Conjugated Secondary Antibody. The BPDE-DNA adduct content in an unknown sample is determined by comparing with a standard curve that is prepared from predetermined BPDE-DNA standards.
Brand OxiSelect™
Sample Type DNA samples
Analytical Method Quantitative
Detection Method Colorimetric
Characteristics The OxiSelect™ BPDE DNA Adduct ELISA Kit is an enzyme immunoassay developed for rapid detection of BPDE-DNA adducts. The quantity of BPDE adduct in DNA samples is determined by relative comparison of a known BPDE-DNA standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.
Components
  1. DNA High-Binding Plate : One 96-well strip plate.
  2. DNA Binding Solution : One 6 mL bottle.
  3. Anti-BPDE Antibody (1000X) : One 20 μL vial of anti-BPDE-I antibody.
  4. Secondary Antibody, HRP Conjugate (1000X) : One 50 μL vial.
  5. Assay Diluent : One 50 mL bottle.
  6. 10X Wash Buffer : One 100 mL bottle.
  7. Substrate Solution : One 12 mL amber bottle.
  8. Stop Solution (Part. No. 310808): One 12 mL bottle.
  9. Reduced DNA Standard : One 200 μL vial of 0.2 mg/mL reduced DNA in TE Buffer.

Box 2 (shipped on blue ice packs)

Material not included
  1. DNA samples such as cell or tissue genomic DNA
  2. DNA Extraction Kit
  3. 1X PBS
  4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  6. Multichannel micropipette reservoir
  7. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length) 3
Background Polycyclic aromatic hydrocarbons (PAHs) are potent, ubiquitous atmospheric pollutants commonly associated with oil, coal, cigarette smoke, and automobile exhaust fumes. Some PAH compounds are also found in cooked foods (e.g. grilled meat, smoked fish) and have been identified as mutagenic and carcinogenic. The toxicity of some PAHs has been demonstrated to induce malignant tumors in animal models and is also commonly believed to significantly contribute to human cancers. One PAH compound, benzo(a)pyrene, is notable for being the first chemical carcinogen to be discovered. Benzo(a)pyrene is a five-ring PAH known to be a procarcinogen, its mechanism of carcinogenesis is dependent on a 3-step enzymatic metabolism (Fig. 1 below) to the final mutagen benzo(a)pyrene diol epoxide (BPDE). Very reactive, BPDE binds covalently to proteins, lipids, and DNA (guanine residues) to produce BPDE adducts. If left unrepaired, DNA adducts may lead to permanent mutations resulting in cell transformation and ultimately tumor development. Figure 1: Benzo(a)pyrene catalyzed to various metabolites by Cytochrome P450 enzymes (CYP) and epoxide hydrolase (EH), resulting in the final carcinogen BPDE.
Comment

  • For detection and quantitation of benzo(a)pyrene diol epoxide (BPDE) DNA adducts
  • BPDE-DNA standard included
  • Suitable for use with DNA samples such as cell or tissue genomic DNA

Plate Uncoated
Reagent Preparation

1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity. Anti-BPDE-I Antibody and Secondary Antibody: Immediately before use dilute the Anti-BPDE-I antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Assay Procedure
  1. Extract DNA from cell or tissue samples using a commercial DNA Extraction kit or other desired method.
  2. Dilute DNA samples to 4 μg/mL in 1X TE Buffer. Note: Samples with high concentrations of BPDE may be further diluted 2-4 fold in 4 μg/mL Reduced BSA. A titration may be performed to ensure the samples fall in the range of the standard curve.
  3. Add 50 μL of unknown DNA samples or BPDE-DNA standards to the wells of the DNA High- Binding plate.
  4. Add 50 μL of DNA Binding Solution to each well. Mix well by pipetting and incubate at room temperature overnight on an orbital shaker. Each DNA sample including unknown and standard should be assayed in duplicate.
  5. Remove the DNA solutions and wash twice with PBS. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1 hour at room temperature. 4
  6. Remove the Assay Diluent. Blot plate on paper towels to remove excess fluid.
  7. Add 100 μL of the diluted Anti-BPDE-I Antibody to all wells and incubate for 1 hour at room temperature on an orbital shaker.
  8. Wash 5 times with 250 μL of 1X Wash Buffer 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.
  9. Add 100 μL of the diluted Secondary Antibody-HRP Conjugate to all wells and incubate for 1 hour at room temperature on an orbital shaker. Wash the strip wells 5 times according to step 8 above.
  10. Warm Substrate Solution to room temperature. Add 100 L 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.
  11. Stop the enzyme reaction by adding 100 μL of Stop Solution to each well. Results should be read immediately (color will fade over time).
  12. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length. Use the Reduced DNA Standard as an absorbance blank.
Restrictions For Research Use only
Handling Advice Avoid multiple freeze/thaw cycles.
Storage 4 °C/-20 °C
Storage Comment Upon receipt, aliquot and store the Reduced DNA and BPDE-DNA Standards at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C until their expiration dates. 3
Supplier Images
ELISA image for BPDE DNA Adduct ELISA Kit (ABIN2345020) BPDE-DNA Standard Curve.
Product cited in: Peters, Wickstrom, Siciliano: "Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?" in: Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals, Vol. 21, Issue 3, pp. 283-91, 2016 (PubMed).

Beranek, Fiala, Kremlacek, Andrys, Hamakova, Chmelarova, Palicka, Borska: "Genetic polymorphisms in biotransformation enzymes for benzo[a]pyrene and related levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts in Goeckerman therapy." in: Toxicology letters, Vol. 255, pp. 47-51, 2016 (PubMed).

Coric, Simic, Pekmezovic, Basta-Jovanovic, Savic Radojevic, Radojevic-Skodric, Matic, Dragicevic, Radic, Bogdanovic, Dzamic, Pljesa-Ercegovac: "Combined GSTM1-Null, GSTT1-Active, GSTA1 Low-Activity and GSTP1-Variant Genotype Is Associated with Increased Risk of Clear Cell Renal Cell Carcinoma." in: PLoS ONE, Vol. 11, Issue 8, pp. e0160570, 2016 (PubMed).

Su, Zhao, Guo, Bin, Yang, Liu, Han, Niu, Ke, Wang, Geng, Jin, Dai, Lin: "Interaction of benzo[a]pyrene with other risk factors in hepatocellular carcinoma: a case-control study in Xiamen, China." in: Annals of epidemiology, Vol. 24, Issue 2, pp. 98-103, 2014 (PubMed).

Barhoumi, Mouneimne, Chapkin, Burghardt: "Effects of fatty acids on benzo[a]pyrene uptake and metabolism in human lung adenocarcinoma A549 cells." in: PLoS ONE, Vol. 9, Issue 3, pp. e90908, 2014 (PubMed).

Chiu, Yen, Tsai, Yang, Liu: "Low-dose benzo(a)pyrene and its epoxide metabolite inhibit myogenic differentiation in human skeletal muscle-derived progenitor cells." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 138, Issue 2, pp. 344-53, 2014 (PubMed).