IM­KOGNW 050915E

JaICA

FOR RESEARCH USE ONLY

Japan Institute for the Control of Aging

Catalog No. KOG­200S/E“New 8­OHdG Check” INSTRUCTIONSThe 8­OHdG Check is a competitive in vitro enzyme­linked immunosorbent assay (ELISA) for quantitative
detection of the oxidative DNA adduct 8­hydroxy­2’­deoxyguanosine (8­OHdG) . Suitable for urine, serum,plasma, tissue and other biological samples. For research use only. Not for diagnostic or medical use.
Read entire insert before use.

1. Kit Contents①8­OHdG Microtiter Plate②Primary Antibody③Primary Antibody Solution④Secondary Antibody⑤Secondary Antibody Solution⑥Chromatic Solution⑦Diluting Solution⑧Washing Solution(5x)⑨Reaction Terminating Solution⑩Standard 8­OHdG Solution⑪Plate Seal

：Precoated with 8­OHdG(8×12wells, split type)1 plate：Anti 8­OHdG Monoclonal antibody (clone N45.1)
1 vial：Phosphate buffered saline
1 vial (6mL)：HRP­conjugated anti mouse antibody
1 vial：Phosphate buffered saline
1 vial (12mL)：3,3',5,5'­tetramethylbenzidine
1 vial (0.25mL)：Hydrogen peroxide/citrate­phosphate buffered saline1 vial (12mL)：5 times concentrated phosphate buffered saline
2 vials (26mLx2)：1M Phosphoric acid
1 vial (12mL)：Purified 8­OHdG (0.5, 2, 8, 20, 80, 200 ng/mL)
1 vial each
2 sheets

All reagent should be stored at 2­8°C. The expiry date is 12 months after manufacture.
After the seal of above contents are opened, this kit should be used within 2 weeks.

2. Additional Materials and Equipments Required①Distilled water.
②50µL micropipettor and pipette tips
③8­channel micropipettor (50～200µL) and pipette tips
④Reagent trays for 8­channel micropipettor⑤A 37°C incubator⑥Microtiter plate reader (measuring wavelength = 450 nm)

3. Priciples of the Procedure①The anti­8­OHdG monoclonal antibody and the sample or standard are added tothe microtiter plate which has been precoated with 8­OHdG. The 8­OHdG
monoclonal antibody reacts competitively with the 8­OHdG bound on the plateand the 8­OHdG in samples solution. Therefore higher concentrations of8­OHdG in the sample solution lead to a reduced binding of the antibody to the8­OHdG on the plate.
②The antibodies which are bound to the 8­OHdG in the sample are washed awayfrom the antibodies that have bound to the 8­OHdG coated on the plate.
③An enzyme­labeled secondary antibody, which is added to the plate, binds to themonoclonal antibody which is bound to the 8­OHdG coated on the plate.
④Unbound HRP­conjugated secondary antibody is removed by washing.
⑤ Addition of the substrate solution results in the development of color inproportion to the amount of anti 8­OHdG antibody bound to the plate.
⑥The reaction is terminated by phosphoric acid, and absorbance at 450 nm is
measured.

4. Assay Procedure
Bring all reagents and samples to room temperature (20­25ºC) before use.
A) Reconstitute the ②Primary Antibody with the ③Primary Antibody Solution.
B) Add 50µL of sample or ⑩Standard per well, as shown in plate diagram.
C) 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.
D) Mix 1 volume of ⑧Washing solution(5x) with 4 volumes of distilled water.
E) 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.
F) Reconstitute the ④Secondary Antibody with the ⑤Secondary Antibody Solution.
G) 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 37°C for 1 hour.
H) At the end of the incubation period, repeat washing as in step E.
I) Prepare substrate solution. Add 1 volume of ⑥Chromatic Solution to 100 volumes of ⑦Diluting 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.
J) Add 100µL of the ⑨Reaction Terminating Solution. Shake the plate from side to side and mix fully.
K) Measure the absorbance at 450 nm using mictotiter plate reader.
Use a standard curve to determine the amount of 8­OHdG present in test samples. Generate thestandard curve by plotting absorbance vs. log (concentration of standards). Then use the absorbancevalues obtained for the test samples to determine the concentrations.
Notices1) Sample Pretreatment
To assay properly, please pre­treat samples as follows. Avoid repeated freeze and thaw.
① Urine : If it's clear, pretreatment is not needed.
Centrifugation at 2,000 ~ 5,000g for 10 ~ 15 minutes is recommended for opaque samples only.
② Serum : Blood samples must be separated to serum immediately. To separate interfering substances,filtration of serum using an ultra filter (cut off molecular weight 10,000) is necessary. Pre­treat ultra
filter following to the maker’s manuals. In order to reduce deviation, diluting samples by more thantwice, while paying attention to concentration range is suggested.
③ DNA in Tissue : It’s necessary to extract and digest DNA in the samples beforehand.
2) Measurement① Strict Control of Incubation Temperature
Measured values may be very much affected with the incubation temperatures, particularly during
primary antibody reaction period. Please pay attention to followings;
a. Try to keep uniform temperature inside a plate constantly.
b. It's recommendable to use water bath rather than dry incubators for the incubation.
② Adjustment of pH for samples
It is necessary to maintain pH of a sample mixed with primary solution between 6.0 to 8.0. It's
recommendable for abnormal urine samples to be diluted with PBS by three times.
③ Thoroughly Washing of Micro plates
It's recommendable to throw the micro plate down on clean paper towel to remove solution in sidewells, after a plate is turn over and solution in side wells is discarded.
④ Cleaning of Instruments and Vessels
Instruments and vessels (such as tips, trays for 8 channel pipettor) to be used, must be clean. If suchtools are used repeatedly, please boil or steep them into alkaline cleanser, then wash thoroughly anddry them before use.
3) Split Usage① Remained parts of kit (plate and reagents) must be kept in a refrigerator and must be used within twoweeks after opened.
② Plates and reagents except chromatic solution are taken out from refrigerator and are kept in room
temperature beforehand. Necessary volume of Chromatic solution may be added to adequate volume
only of Diluting solution just before the reaction. Keep it in the dark.

4)

Use of wells① To avoid edge effects, the use of outer most wells is not recommended. To maintain the uniform
temperature within the wells, please full same volume of solutions or water to the unused wells.
② Blank wells : at the operation of step C, those wells which are not added the reconstituted primary
antibody, will serve as blank wells.
③ The figure below shows a typical layout for sample loading in triplicates for each sample. Wells
indicated with a cross­mark (×) in A and H lines are not used. With this layout, a maximum of 18samples can be assayed in a plate.
1ABCDEFGH

34567Blank (×3)
Standard 0.5 ng/mL
Standard 2 ng/mL
Standard 8 ng/mL
Standard 20 ng/mL
Standard 80 ng/mL
Standard 200ng/mL×××2××Sample­1Sample­2Sample­3Sample­4Sample­5Sample­6××××

5. Standard Curve×910××Sample­7Sample­8Sample­9Sample­10Sample­11Sample­12×××8×1112××Sample­13Sample­14Sample­15Sample­16Sample­17Sample­18×××Absorbance at 450 nm

2 .01 .61 .20 .80 .40 .00 .1

1101001000

8 ­O H d G (n g /m l)

6. References
1. S.Okamoto, and H.Ochi Chemical Abst. 129859a (1992)
2. H.Kasai, P.F.Crain, Y.Kuchino, S.Nishimura, A.Ootsuyama, and H.Tanooka Carcinogenesis 7, 1849­1851(1986)
3. S.Toyokuni, T.Tanaka, Y.Hattori, Y.Nishiyama, A.Yoshida, K.Uchida, H.Hiai, H.Ochi, and T.Osawa
Lab.Invest. 76, 365­374 (1997)
4. M.D.Evans, M.S.Cooke, I.D.Podmore, Q.Zheng, K.E.Herbert, and J.Lunec Discrepancies in themeasurement of UVC­induced 8­oxo­2'­deoxyguanosine: Implications for the analysis of oxidative DNA
damage. Biochemical and Biophysical Research Communications 259 pp374­378 (1999)
5. 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) pp1030­1035 (2000)
6. 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.
JPhysiol Anthropol 19(6) pp287­289 (2000)
7. H.Ochi, M.Hashimoto, J.Kurashige Assessment of functional tea in human using oxidative stress profiletechnique. Proceedings of 2001 International Conference on O­Cha (tea) Culture and Science (2001)
8. M.S.Cooke, M.D.Evans and J.Lunec DNA repair: insights from urinary lesion analysis. Free RadicalResearch 36(9) pp929­932 (2002)
9. 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
8­OHdG. Free Radical Research 36(2) pp189­193 (2002)

7. Assay Flowchart
Sample Pretreatment
Preparation ofPrimary Antibody②Primary Antibody③Primary Antibody Solution

Add sample or ⑩standards 50µL/well

Incubation (Primary antibody reaction)
50µL / well
37℃ for 1 hourPreparation ofSecondary Antibody④Secondary Antibody⑤Secondary Antibody Solution

Preparation of
Washing solution⑧Washing solution (5x) : 25mL
Distilled water:100mL

Wash the Plate
250 µL / well for 3 times

100µL / well
Incubation (Secondary antibody reaction)
37°C for 1 hourPreparation of
Substrate solution⑥Chromatic Solution: 120μL⑦Diluting Solution: 1 vial

Wash the Plate

250 µL / well for 3 times*Prepare just before use.
Incubation
100µL / well
Room temperature for 15 min in the dark
to develop the light blue color⑨Reaction Terminating
Solution

Termination of reaction
100µL / well

Absorbance Reading
wavelength at 450nm

Calculation of Results

8. Technical support
Please visit our web site http://www.jaica.com/biotech/e/Japan Institute For the Control of Aging (JaICA)
710­1 Haruoka, Fukuroi, Shizuoka, 437­0122 JapanE­mail: biotech@jaica.com
Web site: http://www.jaica.com/biotech/e/Phone: +81­538­49­0125
Fax: +81­538­49­1267©2005 JaICA All rights reserved. Japanese Patent No.3091974. Made in Japan.