8-hydroxy-2′-deoxyguanosine (8-OHdG) ELISA Assay kit:
For Research Use Only
Size: 1 x 96 wells
Sensitivity: 0.5 ng/mL
Dynamic Range: 0.5 – 200 ng/mL
Incubation Time: 3.5 hours
Sample Type: Serum, Urine, Biological Fluids
Sample Size: 50 µL
The Eagle Biosciences 8-hydroxy-2′-deoxyguanosine (8-OHdG) 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) ELISA assay kit is for research use only and not to be used in diagnostic procedures.
Bring all reagents to room temperature before beginning 8-hydroxy-2′-deoxy-guanosine (8-OHdG) 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.
- 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 37°C for 1 hour.
- Measured values may be very much affected with the incubation temperatures, particularly during primary antibody reaction period. It is recommended to use water bath rather than dry incubators for the incubation.
- Mix 1 volume of Washing Solution (5x) with 4 volumes of distilled water.
- Pour off contents of wells 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. The use of washing machines or aspirators is not recommended.
- Reconstitute the Secondary Antibody with the Secondary Antibody Solution.
- Add 100µL of reconstituted secondary antibody per well. Shake the plate from side to side and mix fully. Cover the plate with an adhesive strip. Incubate 37°C for 1 hour.
- At the end of the incubation period, repeat washing as in step 5.
- Prepare substrate solution. Add 1 volume of Chromatic Solution to 100 volumes of Diluting Solution just before use. Add 100µL of substrate solution per well. Shake the plate from side to side and mix fully. Incubate at room temperature for 15 minutes in the dark.
- Add 100µL of the Reaction Terminating Solution. Shake the plate from side to side and mix fully.
- Measure the absorbance at 450 nm using microtiter plate reader.
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)