8-hydroxy 2 deoxyguanosine ELISA Kit

Product details

8-hydroxy 2 deoxyguanosine ELISA Kit (8-OHdG) ab201734 is a competitive ELISA kit to measure 8-Hydroxy-2'-deoxyguanosine with a sensitivity of 0.59 ng/mL.

How the assay works

8-hydroxy 2 deoxyguanosine ELISA Kit ab2017341 is a competitive ELISA. There are 2 types of competitive ELISAs:

• antigen down: The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody. The reference antigen is pre-coated on a multi-well plate. The sample is added to the plate wells with a labeled antibody. Depending on the amount of antigen in the sample, more or fewer free antibodies will be available to bind the reference antigen. This means that the more of the antigen there is in the sample, the less reference antigen will be detected and the weaker the signal will be.
• antibody down: the labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody, which is immobilized on the plate. The lower the amount of antigen in the sample, the stronger the signal due to more labelled antigen in the well.

Assay specificity

Our ELISA kits are rigorously validated to ensure the highest level of consistency and reproducibility.

8-hydroxy 2 deoxyguanosine ELISA Kit (8-OHdG) ab201734 protocol summary

- Add 50 µL of Standards (S1–S8), Samples, or Blank components to the appropriate wells.
     - Standards/Samples: 50 µL of prepared standard or sample + 50 µL Antibody Preparation
     - Zero Standard: 50 µL Zero Standard + 50 µL Antibody Preparation
     - Blank: 50 µL Standard & Sample Diluent + 50 µL Antibody Diluent
- Cover plate and incubate 1 hour at room temperature (20–25°C).
- Wash the plate 4 times with 1X Wash Buffer (300 µL per wash).
- Add 100 µL TMB Substrate to each well.
- Cover and incubate 30 minutes at room temperature in the dark.
- Add 100 µL Stop Solution to each well. Mix gently.
- Read absorbance at 450 nm within 30 minutes.

It is important to note that the 8-OHdG antibody used in this assay recognizes both free 8-OHdG and DNA-incorporated 8-OHdG. Since complex samples such as plasma, cell lysates, and tissues are comprised of mixtures of DNA fragments and free 8-OHdG, concentrations of 8-OHdG reported by ELISA methodology will not coincide with those reported by LC-MS where the single nucleoside is typically measured. This should be kept in mind when analyzing and interpreting experimental results.

8-hydroxy-2-deoxy Guanosine (8-OHdG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker of oxidative stress. Hydroxylation of guanosine occurs in response to both normal metabolic processes and a variety of environmental factors (i.e., anything that increases reactive oxygen and nitrogen species). Increased levels of 8-OHdG are associated with the aging process as well as with a number of pathological conditions including cancer, diabetes, and hypertension.

In complex samples, such as plasma, cell lysates, and tissues, 8-OHdG can exist as either the free nucleoside or incorporated in DNA. Once the blood enters the kidney, free 8-OHdG is readily filtered into the urine, while larger DNA fragments remain in the bloodstream. Because of the complexity of plasma samples, urine is a more suitable matrix for the measurement of free 8-OHdG than plasma. Urinary levels of 8-OHdG range between 2.7- 13 ng/mg creatine, while plasma levels of free 8-OHdG have been reported to be between 4-21 pg/mL as determined by LC-MS.