1、Animals applicable

Q: What kind of animals is applicable to 8-OHdG ELISA?

A: Applicable to urine samples from human, mouse, rat, rabbit, dog, cattle, horse, etc. 8-OHdG Check ELISA kit can detect 8-OHdG in urine, serum, plasma, blood cells, tissue and cultured cells. There are two types of 8-OHdG ELISA products with different assay ranges. For urine samples, "New 8-OHdG Check" is suitable. For serum and tissue samples, we recommend to apply to "Highly Sensitive 8-OHdG Check" after pretreatment of samples.

The sample pretreatment procedure may be different depending on the type of samples. Please confirm the pretreatment procedure for your samples. If you are planning to measure 8-OHdG in urine samples, please remove cells and insluble materials from urine before application to ELISA.

2、Sample dilution

Q: What should be used for sample dilution?

A: Phoshate buffered saline (PBS, pH7.4) is recommended. If urine sample is from healthy human, sample can be applied to "New 8-OHdG Check ELISA" as it is, but if urine sample is from patients, we recommend to dilute samples for 2 or 4 times with PBS.

3、Stability of urinary 8-OHdG

Q: How urine samples should be stored?

A: 8-OHdG is known to chemically stable. Urine sample can be stored for 3 days at room temperature, for 7 days at 4 °C. But please take care for contamination of bacteria. If hydrochloric acid is used to prevent bacterial growth, please adjust pH before apply to ELISA.

Urine samples can be stored for longer period, for years at -80 °C. But please avoid repeated freeze / thaw cycle. When frozen urine sample is thaw, insolbule materials may be observed in some cases. Please remove insoluble materials by centrifuge before application to ELISA.

According to the recent paper, urine samples are stable for at least 2 years if stored at below -80 °C.

Y Matsumoto, et.al., J Occup Health 50, p366-372 (2008)

4、Type of samples applicable

Q: What kind of samples are applied to 8-OHdG ELISA?

A: In addition to urine samples, serum / plasma, ascite samples, outer liquid of artificial dialysis and saliva samples are known to be applied to 8-OHdG ELISA. Pretreatment is required for these samples. DNA samples isolated from blood cells, sperm, organs and cultured cells can be applied to 8-OHdG ELISA.

[Assay example for saliva]

New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals. M Takane, et. al., J Periodontol.73(5), p551-554 (2002)

5、8-OHdG concentration in normal human urine:

A) Mean concentration: 13.6 ng/mL (250 males) and 10.3 ng/mL (250 females)

B) 8-OHdG extretion ratio: 8.6 ng/kg/h (250 males and 250 females)

C) Creatinin correction: 8.4 ng/mg CRE (250 males and 250 females)

6、Urine sampling and correction:

8-OHdG concentraion in random urine is not stable, depending on individual variation, the time in the day of sample collection, sex and some other factors. To obtain the best result, lease take care for sampling and correction methods.

About creatinine correction:

Creatinie correction is known to be a good method to obtain stable data. But it is not suitable for some researchers who are interested in the effect of exercise to oxidative stress. That's because creatinine concentration will be increased by exercise.

1) Effect of repeated exercise on urinary 8-hydroxy-deoxyguanosine excretion in humans. Free Radic Res. 1997 Jun;26(6):507-14. Okamura K, et. al.

In this paper, effects of running are assessed using 8-OHdG excretion per day. A significant increase of urinary 8-OHdG is observed (from 265.7+/-75.5 to 335.6+/-107.4 pmol/ kg/ day).

2) Habitual long-distance running does not enhance urinary excretion of 8-hydroxydeoxyguanosine. Eur J Appl Physiol Occup Physiol. 1997;75(5):p467-469. Pilger A,et. al.

In this paper, effects of running are assessed by urinary 8-OHdG with creatinine correction. Both 8-OHdG and creatinine concentration increased. But the effect of running to 8-OHdG level is not significant (0.12 - 6.45 µmol/mol CRE).

7、Trouble shootings:

Please clice HERE to see technical tips for New 8-OHdG Check ELISA kit.

8、Serum / plasma samples:

To detect 8-OHdG in serum and plasma samples, pretreatment is required. Please remove proteins by ultrafiltration, and apply to "Highly Sensitive 8-OHdG Check". For more information, click HERE.

9、Blood collection tubes:

Blood collection tubes are commercially available. For example, NIPRO code.30-122 is suitable. Please note that serum 8-OHdG concentration may be increased if whole blood is placed for hours at room temperature. We recommend to start centrifuge in 20 minutes after blood collection. Serum samples must be stored below -20 °C.

10、Human serum 8-OHdG:

8-OHdG concentration in male serum is higher than that of female. Serum 8-OHdG is higher in smokers than in non-somokers.

Reference:The relationship between smoking habits and serum levels of 8-OHdG, oxidized LDL antibodies, Mn-SOD and carotenoids in rural Japanese residents., J Epidemiol 2003;13(1):p29-37.Suzuki K, et.al.

11、8-OHdG in tissue samples:

After the hydrolysis of DNA extracted from tissues or cells, 8-OHdG levels can be measured using "Highly Sensitive 8-OHdG Check".

Reference:Coexposure to benzo[a]pyrene plus ultraviolet A induces 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in human skin fibroblasts: preventive effects of anti-oxidant agents. Environmental Toxicology and Pharmacology 12, p37-42, 2002

12、Extraction of DNA:

In order to measure 8-OHdG in tissue samples or cultured cells, DNA must be extracted by NaI method. NaI-based kits for DNA extractions are commercially available. For example,

DNA extractor® TIS kit (WAKO Chemicals, code. 296-67701) may be useful.

References:

ML Hamilton, et.al. A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.

Nucleic Acid Res. 29(10), p2117-2126(2001)

HJ Helbock, et. al. DNA oxidation matters: The HPLV-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine.

Proc Natl Acad Sci USA 95,p288-293(1998)

13、The sample volume required to

measure 8-OHdG in tissue or cells:

Q: What is the required sample volume to measure 8-OHdG in tissue or cells?

A: This depends on the experiment, however, usually 8-OHdG exists between 10-6 and 10-5 per dG in healthy human tissue. 8-OHdG can be measured between 0.125-10ng/mL using the "Highly Sensitive 8-OHdG Check", therefore in order to detect 8-OHdG certainly we recommend prepareing 200 µg/135µL of DNA.

14、Nuclease P1 reagent:

You may be able to find nuclease P1 enzyme from local agency for biochemical reagents. In addition, nuclease P1 is available at our web site.

Code: OPNUCP1, Nuclease P1 from Penicillium citrinum: 25,000 JPY + freight.

15、About Argon substitution:

Q: What does "Argon substitution for detection of 8-OHdG in tissue DNA" mean?

A: Argon substitution is to replace air (containing oxygen) with argon gas. That's because purified DNA may be oxidized in the presence of oxygen. To prevent the possibility of oxidization during sample preparation, we recommend performing "argon substitution" for reagents and samples. Argon gas is gently blown into the test tube containing the sample solution or reagents. However if the experiment will be carried out immediately, argon substitution will not be necessary.

Reference:CG Fraga, et. al.: Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci USA 87, p4533-4537, 1990.

16、Pretreatment of tissue samples:(What is Chelex® treatment?)

It is reported that DNA can be oxidized by the Fenton reaction in the presence of trace divalent metal ions. Divalent metal ions can be removed by Chelex® resin (Chelex® is a registered trademark of BioRad Laboratories). For reliable results, Chelex® -treated water should be used to detect 8-OHdG in DNA samples.

17、Can't find Ultrafree-MC:

As far as we know Ultrafree-MC (Millipore corporation, USA, catalog #UFC3LGC) is out of production. As an alternative, Microcon YM-10 (Millipore, catalog#42407) can be used.

18、Anatlytic testing service:

JaICA is a registered medical testing institute in Japan, and is able to accept your urine and serum samples.

Testing service is also available at Genox corporation (Baltimore, USA).

Please take care that the testing service is for research use only. Not applicable for medical, diagnostic or other use.

19、Number of samples measured:

Q:How many samples can be measured using 1 kit?

A:If experiment will be conducted in N=3, 18 samples can be measured (total 54 assays). One ELISA kit contains a 96-wells micro titer plate.

In some cases, at the top and bottom row of wells, error of variance from the “edge effect” can be observed. Although all wells are prepared uniformly to obtain reliable data, it is recommended not to use wells in top and bottom rows. 6 levels by 3 wells (total 18 wells) will be used for standards. As a result, 54 wells can be used for samples.

20、How to calculate the results:

Q: What algorithms are suitable for the standard curve?

A: Any algorithm function can be used alternatively to calculate the results if it is an approximate semi-logarithm curve (Absorbance=Antilogarithm, Concentration=Logarithm). Especially since recent micro plate readers have useful functions to draw the standard curves, you can use it. The semi-logarithm line graph is also enough to use as an approximate function.

If you don't have appropriate software, please feel free to send us your law data by e-mail entitled "Calibration of ELISA kit". We will convert your raw data to 8-OHdG concentration.

21、Partial use of the ELISA kit:

Q: How many times can the ELISA kit be partially used?

A: The ELISA kit is made up for 8 wells * 12 columns, therefore can be divided accordingly to meet the number of samples being measured. Please be aware that a new calibration curve is necessary for each measurement of samples. For a calibration curve 6 levels* 3=18 wells are needed. Almost all samples remain stable when stored at lower than -20°C. Therefore more samples can be measured when you test all of them at the same time.

22、Assay example for ovarian endometrioma:

To detect 8-OHdG in ovarian endometrioma solution, please remove viscous components by centrifuge or filtration, and subsequently remove proteins by ultrafilration as the same protocol as that for serum samples.

Ovarian endometrioma: 0.58 ng/mL 8-OHdG

Serous Cystadenoma: 0.06 ng/mL 8-OHdG

Mucinous cystadenoma: 8-OHdG not detected.

Reference: S Fujii, 31th meeting of Japan BioIron Sociery, Abstruct in Japanese p9-10 (2007)

23、Which micro plate reader to be used?

Any micro plate reader with 450nm filter may be suitable.

24、Protocol to hydrolyse DNA using DNase I:

Protocols are suggested in following papers:

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry.Nucleic Acids Res 29(3) E12(2001).Dizdaroglu M, Jaruga P, Rodriguez H

Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid. Mol Vis 14 p644-651(2008)Wang AL, Lukas TJ, Yuan M, Neufeld AH.

25、Reference wavelenghth: 600 - 650 nm would be useful.

26、Feature of our 8-OHdG ELISA:

The monoclonal antibody (clone N45.1) which is used for 8-OHdG Check ELISAs is highly specific to 8-OHdG. It is reported that cross reactivity to 8-OHdG analogues (guanosine(G),7-methyl-G, 6-SH-G, 8-Bromo-G, dA, dC, dT, dI, dU, dG, O6-methyl-dG, 8-OHdA, guanine(Gua),O6-methyl-Gua, 8-OH-Gua, creatinine, 8-sulfhydryl-G, 8-OH-G) is less than 1% in comparison with that to 8-OHdG.

[Reference] S.Toyokuni, et.al.: Quantitative immunohistochemical determination of 8-hydroxy-2'deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab. Invest. 76(3), p365-374 (1997)

27、Creatinine determination:

Adjustment by creatinine concentration is useful for assessment of 8-OHdG in urine samples. Creatinine assay kits are commercially available.

LabAssay™ Creatinin, Code. 290-65901, WAKO Chemical Co.Ltd.

This kit can be applied to human and animal urine.