BPDE Protein Adduct ELISA Kit

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Antigen
Reactivity
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Method Type
Competition ELISA
Application
ELISA
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Purpose BSA standards or protein samples (10 μg/mL) are adsorbed onto a 96-well plate for 2 hrs at 37°C. The BPDE-protein adducts present in the sample or standard are probed with an Anti-BPDE-I Antibody, followed by an HRP Conjugated Secondary Antibody. The BPDE protein adduct content in an unknown sample is determined by comparing with a standard curve that is prepared from predetermined BPDE-BSA standards.
Brand OxiSelect™
Sample Type Cell Lysate, Serum, Plasma
Analytical Method Quantitative
Detection Method Colorimetric
Characteristics The OxiSelect™ BPDE Protein Adduct ELISA Kit is an enzyme immunoassay developed for rapid detection of BPDE-protein adducts. The quantity of BPDE adduct in protein samples is determined by relative comparison of a known BPDE-BSA standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.
Components
  1. 96-well Protein Binding Plate : One strip well 96-well plate.
  2. Anti-BPDE Antibody (1000X) : One 20 μL vial of anti-BDPE-I antibody.
  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. Reduced BSA Standard : One 500 μL vial of 1 mg/mL reduced BSA in PBS.

Box 2 (shipped on blue ice packs)

Material not included
  1. Protein samples such as purified protein, plasma, serum, cell lysate
  2. 1X PBS
  3. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  5. Multichannel micropipette reservoir
  6. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
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) protein adducts
  • BPDE-BSA standard included
  • Suitable for use with protein samples such as purified protein, plasma, serum, or cell lysates

Plate Uncoated
Reagent Preparation
  • 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate 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. 3 Preparation of Standard Curve 1. Freshly prepare 10 μg/mL of Reduced BSA by diluting the 1 mg/mL BSA standard in 1X PBS. Example: Add 50 μL to 4.95 mL of 1X PBS. 2. Freshly prepare 1 μg/mL of BPDE-BSA by diluting the 1 mg/mL BPDE-BSA standard in 10 μg/mL of Reduced BSA. Example: Add 2 μL to 2.0 mL of 10 μg/mL Reduced BSA. 3. Prepare a series of BPDE-BSA standards according to Table 1. 1 BPDE-BSA 10 Reduced BSA Final BPDE-Standard Tubes μg/mL μg/mL BSA Conc. (μL) (μL) (ng/mL) 1 1000 0 1000 2 500 of tube #1 500 500 3 500 of tube #2 500 250 4 500 of tube #3 500 125 5 500 of tube #4 500 62.5 6 500 of tube #5 500 31.3 7 500 of tube #6 500 15.6 8 0 500 0 Table 1. Preparation of BPDE-BSA Standard Curve
Sample Preparation
  1. Perform a protein assay such as Bradford or BCA on all samples to determine the protein concentration. Note: Cell and tissue lysates should not be prepared in lysis buffer containing Triton X-100, NP- 40, or Igepal CA-630 because these detergents interfere with protein coating of the plate unless the detergent concentration in the 10 μg/mL protein samples is no more than 0.001 % . We recommend lysis by homogenization or sonication.
  2. Dilute each protein sample to 10 μg/mL in 1X PBS prior to use in the assay.
Assay Procedure
  1. Prepare unknown samples according to the Preparation of Samples section above. Each 10 μg/mL protein sample and BSA Standard should be assayed in duplicate or triplicate.
  2. Add 100 μL of the 10 μg/mL protein samples or Reduced/BPDE-BSA standards to the 96-well Protein Binding Plate. Incubate at 37 °C for at least 2 hours or 4 °C overnight.
  3. Wash wells 2 times with 250 μL 1X PBS per well. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess wash solution. 4
  4. Add 200 μL of Assay Diluent per well and incubate for 1-2 hours at room temperature on an orbital shaker.
  5. Wash 3 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.
  6. 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. Wash the strip wells 3 times according to step 5 above.
  7. 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 5 above.
  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 to each well. Results should be read immediately (color will fade over time).
  10. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length. Use the Reduced BSA Standard as an absorbance blank. 5
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 BSA and BPDE-BSA Standards at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C.
Supplier Images
ELISA image for BPDE Protein Adduct ELISA Kit (ABIN2344942) BPDE-BSA Standard Curve.
Product cited in: Lai, Lee, Bai, Yang, Chuang, Wu, Chuang: "Protein oxidation and degradation caused by particulate matter." in: Scientific reports, Vol. 6, pp. 33727, 2016 (PubMed).

Feng, Huang, Chuang, Chen, Lee, Ho, Bien, Yang, Chuang: "Dysfunction of methionine sulfoxide reductases to repair damaged proteins by nickel nanoparticles." in: Chemico-biological interactions, Vol. 236, pp. 82-9, 2015 (PubMed).