For research use.

IM­KOGHS 040914E“Highly Sensitive 8­OHdG Check” INSTRUCTIONSThe 8­OHdG ELISA kit is a competitive in vitro enzyme­linked immunosorbent assay for quantitative measurement ofthe oxidative DNA adduct 8­hydroxy­2’­deoxyguanosine (8­OHdG) in tissue, urine etc.

1. Kit Contents①8­OHdG Microtiter plate: precoated with 8­OHdG (8×12wells, split type)②Primary antibody: monoclonal antibody specific for 8­OHdG③Primary antibody solution : phosphate buffered saline④Secondary antibody: HRP­conjugated antibody⑤Secondary antibody solution: phosphate buffered saline⑥Chromatic solution: 3,3',5,5'­tetramethylbenzidine⑦Diluting solution: hydrogen peroxide/citrate­phosphate buffered saline⑧Washing solution(×5): 5 times concentrated phosphate buffered saline**⑨Reaction terminating solution: 1M phosphoric acid
⑩Standard 8­OHdG solution : 0.125, 0.25, 0.5, 1, 4, 10 ng/ml 1vial each
⑪Plate seal

1 plate1 vial1 vial (ca.6ml)
1 vial1 vial (ca.12ml)
1 vial (ca.0.25ml)
1 vial (ca.12ml)
2 vials (ca.26ml×2)
1 vial (ca.12ml)
total 6 vials (ca.1ml)
2 sheets*All reagent should be refrigerated at 2­8℃.
*The kit should not be used beyond 9 months past the manufacturers date stamped on the exterior of the box.
**Dilute ⑧Washing solution by 5 times (v/v) with distilled water for use.

2. Additional Materials and Equipments Required①50µl pipettor and tips
②8­channel micropipettor and tips（50­200µl）③Trays for 8­channel pipettor④Refrigerator⑤Microplate reader (450 nm)

3. Summary of Assay Procedure①The 8­OHdG monoclonal antibody and the sample or standard are added to the microtiterplate which has been precoated with 8­OHdG. The 8­OHdG monoclonal antibody reacts
competitively with the 8­OHdG bound on the plate and the 8­OHdG in samples solution.
Therefore higher concentrations of 8­OHdG in the sample solution lead to a reduced
binding of the antibody to the 8­OHdG on the plate.
②The antibodies which are bound to the 8­OHdG in the sample are washed away from theantibodies 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 enzyme­labeled secondary antibody is removed by a wash step.
⑤Addition of a chromatic substrate results in the development of color in proportion to theamount of antibody bound to the plate.
⑥The color reaction is terminated and the absorbance is measured.

­1­

4. Assay Procedure
Bring all reagents and samples to room temperature before use (20­25℃), and proceed through the following 10 steps.
① Reconstitute the primary antibody with the primary antibody solution. Allow dissolving completely.
② Add 50µl of sample or standard per well, as shown in plate diagram. To ensure accuracy, do not use outer mostwells.
③ Add 50µl of reconstituted primary antibody per well. Shake the plate from side to side and mix fully. Cover platewith adhesive strip, making sure it is sealed tightly. Incubate at 4℃ for over night.
④ Pour off contents of plate into sink. Pipette 250µl washing solution into each well. After washing thoroughly byshaking the plate from side to side, dispose of washing solution. Invert plate and blot against clean paper towel toremove any remaining washing buffer. Repeat wash two times more.
⑤ Reconstitute the secondary antibody with the secondary antibody solution. Allow dissolving completely.
⑥ Add 100µl of constituted secondary antibody per well. Shake the plate from side to side and mix fully. Cover theplate with an adhesive strip. Incubate room temperature for 1 hour.
⑦ At the end of the incubation period, repeat wash as in step ④.
⑧ 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 atroom temperature for 15 minutes. This incubation should be done in the dark, i.e. shield the plate with aluminum
foil.
⑨ Add 100µl of the reaction terminating solution. Shake the plate from side to side and mix fully.
⑩ After terminating the reaction, read the absorbance at 450 nm.
Use a standard curve to determine the amount of 8­OHdG present in test samples. Generate the standard curve byplotting absorbance vs. log (concentration of standards). Then use the absorbance values obtained for the testsamples to determine the concentrations.
Notices1) Sample Pretreatment
To assay properly, please pre­treat samples desired bellow. In addition, please avoid repeated freeze thaw samples. Inorder to confirm the aptitude of assay methods on a new sample, implementing recovery test of standard 8­OHdG
added into the new sample is recommended.
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 ofserum using an ultra filter (cut off molecular weight 10,000) is necessary. Pre­treat ultra filter following to themaker’s manuals. In order to reduce deviation, diluting samples by more than twice, while paying attention toconcentration 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 much affected by the reacting conditions. Please pay attention to the reacting
conditions.
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 piled paper towel to remove solution in side wells, after aplate 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 such tools areused repeatedly, please boil or steep them into alkaline cleanser, then wash thoroughly and dry them before use.
3) Split Usage① Remained parts of kit (plate and reagents) must be kept in a refrigerator and must be used within two weeks afteropened.
② Plates and reagents except chromatic solution (⑥) are taken out from refrigerator and are kept in room
temperature beforehand. Necessary volume of Chromatic solution may be added to adequate volume only ofDiluting solution (⑦) just before the reaction. Keep it in the dark.

­2­4)Wells Usage① 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 ③, those wells which are not added the reconstituted primary antibody, willserve as blank wells.
③ The figure below shows a typical layout for sample loading in triplicates for each sample. Wells indicated with across­mark (×) in A and H lines are not used. With this layout, a maximum of 18 samples can be assayed in aplate.
1ABCDEFGH

23Blank (×3)
0.125ng/ml(×3)
0.25ng/ml(〃)
0.5ng/ml(〃)
1ng/ml(〃)4ng/ml(〃)10ng/ml(〃)×××4××5×123456×

5. Standard Curve

6×(×3)(〃)(〃)
(〃)
(〃)
(〃)
×

7××8
×7
8
9
10
1112×9×(×3)(〃)(〃)(〃)(〃)(〃)
×10××11×13
1415
16
1718×12×(×3)(〃)(〃)
(〃)
(〃)
(〃)
×1.2

Absorbance at 450 nm

1.00.8
0.6
0.4
0.2
0.0
0.1

18-OHdG (ng/ml)

106. References
1.
2.
3.
4.

5.

6.

7.
8.
9.

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, 1849­1851 (1986)
S.Toyokuni, T.Tanaka, Y.Hattori, Y.Nishiyama, A.Yoshida, K.Uchida, H.Hiai, H.Ochi, and T.Osawa Lab.Invest.
76, 365­374 (1997)
M.D.Evans, M.S.Cooke, I.D.Podmore, Q.Zheng, K.E.Herbert, and J.Lunec Discrepancies in the measurement ofUVC­induced 8­oxo­2'­deoxyguanosine: Implications for the analysis of oxidative DNA damage. Biochemical andBiophysical Research Communications 259 pp374­378 (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) pp1030­1035 (2000)
WenYing Fan, Kazunori Ogusu, Katsuyasu Kouda, Harunobu Nakamura, Tomoaki Satoh, Hirotomo Ochi andHiroichi Takeuchi Reduced oxidative DNA damage by vegetable juice intake: A controlled trial.
J PhysiolAnthropol 19(6) pp287­289 (2000)
H.Ochi, M.Hashimoto, J.Kurashige Assessment of functional tea in human using oxidative stress profile
technique. Proceedings of 2001 International Conference on O­Cha (tea) Culture and Science (2001)
M.S.Cooke, M.D.Evans and J.Lunec DNA repair: insights from urinary lesion analysis. Free Radical Research36(9) pp929­932 (2002)
Tadashi Matsubasa, Takako Uchino, Shinnyo Karashima, Yuichi Kondo, Kenichi Maruyama, Masako Tanimuraand Fumio Endo Oxidative Stress in very low Birth Weight Infants As Measured by Urinary 8­OHdG. Free
Radical Research 36(2) pp189­193 (2002)­3­7. Assay Flowchart
Preparation of Reagent

Sample Pretreatment

primary antibody

Sample/Antibody Incubation

secondary antibody

add sample or standard 50µl
add primary antibody 50µｌ
incubation at 4℃ for over night

Wash the Plate

3 times

add washing solution 250µl

Secondary Antibody Incubation
add secondary antibody solution 100µl
incubation at room temperature for 1 hourPreparation ofEnzyme Substrate

Wash the Plate

3 times

add washing solution 250µl
enzyme substrate

Substrate Incubation
add reconstitution enzyme substrate 100µl
room temperature for 15 min in the dark todevelop the light blue color

Reaction Termination Step
addition of reaction terminating solution 100µlchanges the color of solution to yellow

Absorbance Reading
wavelength at 450nm

Calculation of Results

Japan Institute For the Control of Aging710­1 Haruoka, Fukuroi City, Shizuoka Pref., Japan, 437­0122
E­mail: biotech@jaica.com
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