8-hydroxy-2′-deoxyguanosine (8-OHdG) Ultrasensitive ELISA Assay kit:
For Research Use Only
Size: 1 x 96 wells
Sensitivity: 0.125 ng/mL
Dynamic Range: 0.125 – 10 ng/mL
Incubation Time: Overnight
Sample Type: Serum, Urine, Biological Fluids
Sample Size: 50 µL
The Eagle Biosciences 8-hydroxy-2′-deoxyguanosine (8-OHdG) Ultrasensitive ELISA assay kit is intended for the quantitative determination of adduct 8-hydroxy-2’-deoxy-guanosine (8-OHdG) in urine, serum or biological samples by enzyme linked immunoassay (ELISA). The 8-hydroxy-2′-deoxyguanosine (8-OHdG) Ultrasensitive ELISA assay kit is for research use only and not to be used in diagnostic procedures.
Bring all reagents of the to room temperature before beginning 8-hydroxy-2′-deoxy-guanosine (8-OHdG) Ultrasensitive ELISA assay kit. Determine the number of microwells needed for the assay (each sample, standard, and control should be assayed in duplicate). Bring all reagents and samples to room temperature (20-25ºC) before use.
- Reconstitute the primary antibody with the primary antibody solution. Allow dissolving completely.
- Add 50µl of sample or standard per well.
- Add 50µl of reconstituted primary antibody per well. Shake the plate from side to side and mix fully. Cover plate with adhesive strip, making sure it is sealed tightly. Incubate at 4C for overnight.
- Pour off contents of plate into sink. Pipette 250µl washing solution into each well. After washing thoroughly by shaking the plate from side to side, dispose of washing solution. Invert plate and blot against clean paper towel to remove any remaining washing buffer. Repeat wash two times more.
- Reconstitute the secondary antibody with the secondary antibody solution. Allow dissolving completely.
- Add 100µl of constituted secondary antibody per well. Shake the plate from side to side and mix fully. Cover the plate with an adhesive strip. Incubate room temperature for 1 hour.
- At the end of the incubation period, repeat wash as in step 4.
- Reconstitute the chromatic solution (enzyme substrate solution) with 100 times volume of the diluting solution. Add 100µl of the reconstituted enzyme substrate per well. Shake the plate from side to side and mix fully. Incubate at room temperature for 15 minutes. This incubation should be done in the dark, i.e. shield the plate with aluminum foil.
- Add 100µl of the reaction terminating solution. Shake the plate from side to side and mix fully.
- After terminating the reaction, read the absorbance at 450 nm.
- Use a standard curve to determine the amount of 8-OHdG present in test samples. Generate the standard curve by plotting absorbance vs. log (concentration of standards). Then use the absorbance values obtained for the test samples to determine the concentrations.
Typical Standard Curve:
- S.Okamoto, and H.Ochi Chemical Abst. 129859a (1992)
- H.Kasai, P.F.Crain, Y.Kuchino, S.Nishimura, A.Ootsuyama, and H.Tanooka Carcinogenesis 7, 1849-1851 (1986)
- S.Toyokuni, T.Tanaka, Y.Hattori, Y.Nishiyama, A.Yoshida, K.Uchida, H.Hiai, H.Ochi, and T.Osawa Lab.Invest. 76, 365-374 (1997)
- M.D.Evans, M.S.Cooke, I.D.Podmore, Q.Zheng, K.E.Herbert, and J.Lunec Discrepancies in the measurement of UVC-induced 8-oxo-2′-deoxyguanosine: Implications for the analysis of oxidative DNA damage. Biochemical and Biophysical Research Communications 259 pp374-378 (1999)
- Tomoko Shimoike, Toyoshi Inoguchi, Fumio Umeda, Hajime Nawata, Katsumi Kawano and Hirotomo Ochi The meaning of serum levels of advanced glycosylation end products in diabetic nephropathy. Metabolism 49(8) pp1030-1035 (2000)
- WenYing Fan, Kazunori Ogusu, Katsuyasu Kouda, Harunobu Nakamura, Tomoaki Satoh, Hirotomo Ochi and Hiroichi Takeuchi Reduced oxidative DNA damage by vegetable juice intake: A controlled trial. J Physiol Anthropol 19(6) pp287-289 (2000)
- H.Ochi, M.Hashimoto, J.Kurashige Assessment of functional tea in human using oxidative stress profile technique. Proceedings of 2001 International Conference on O-Cha (tea) Culture and Science (2001)
- M.S.Cooke, M.D.Evans and J.Lunec DNA repair: insights from urinary lesion analysis. Free Radical Research 36(9) pp929-932 (2002)
- Tadashi Matsubasa, Takako Uchino, Shinnyo Karashima, Yuichi Kondo, Kenichi Maruyama, Masako Tanimura and Fumio Endo Oxidative Stress in very low Birth Weight Infants As Measured by Urinary 8-OHdG. Free Radical Research 36(2) pp189-193 (2002)