Home > Protocols > Detection of 8-OHdG in serum/plasma Rev.090309
Detection of 8-OHdG in serum/plasma
Pretreatment is needed for 8-OHdG detection in serum/plasma samples
8-OHdG ELISA kits may be affected by high-molecular weight substances such as proteins. To detect 8-OHdG accurately in serum/plasma or urine which contains proteins, removal of proteins from sample fluid is needed. This is an example for sample pretreatment by ultrafiltration method.
Example
Please prepare ultrafilter with cut-off molecular weight 10kDa, for example Microcon YM-10(catalog#42407, Millipore). If 300 µL of serum is applied to Microcon YM-10 filter, approx. 200 uL of filtrate may be obtained. Please apply the filtrate to Highly Sensitive 8-OHdG Check ELISA. Please take care that 8-OHdG concentration of normal human serum may be low (0.1 - 0.3 ng/mL), and in some cases serum 8-OHdG would be below detection limit ( < 0.125 ng/mL).
Prepare Microcon YM-10 (catalog#42407, Millipore).
Soak YM-10 filter in distilled water for 8-12 hours, and wash it following the instruction manual. Ultra filters other than Microcon YM-10 may be suitable if the molecular cut-off is 10 kDa. Please refer manufacturer's protocol.
Take the filter from water, and remove waterdrop from outside of the filter.
Pour 300 µL of distilled water into the filter unit.
To clean the filter, centrifuge for 20 minutes at 10,000G.
Invert the filter upside down.
Remove waterdrop from the filter by flushing (for example, 3 minutes at 1,000G).
Pour 300 uL of serum or plasma into the filter unit. Please take care not to pour over 300 µL, because in some cases retentate can leak though the side wall of the filter.
Please take care not dry up washed filters.
Centrifuge for 50 minutes at 10,000 G. 150-200 µL of filtrate may be obtained.
The temperature should be kept below 20 °C during the centrifugation.
Apply the filtrate to Highly Sensitive 8-OHdG Check ELISA. If dilution is needed, please use phosphate buffered saline (PBS pH7.4) as diluent.
 
Sampling
After collection of blood, please prepare serum or plasma as soon as possible, because reactive oxygen species (ROS) can be released from activated blood cells. Please start centrifuge in 20 minutes to separate from blood cells.
Serum or plasma samples can be stored at -20 °C. For long term storage, please store at -80 °C. Avoid freeze and thaw cycles. Sample pretreatment should be done in the same day of assay.
Application to other biological fluids (recovery testing)
In almost all cases, there may be no problem on measuring 8-OHdG in serum, plasma and urine samples from normal human, if samples are pretreated correctly as described above. But if you are planning to apply to other biological fluids such as saliva, or apply to samples from animal models, we recommend to test the accuracy by recovery testing.

Recovery testing:
1) Please measure 8-OHdG in following samples.
[base sample]: mix 9 volumes of sample with 1 volume of PBS.
[8-OHdG]: mix 9 volumes with 1 volume of 8-OHdG standard (10 ng/mL).
[mixed sample]: mix 9 volumes of sample with 1 volume of 8-OHdG standard (10 ng/mL).
2) calculate the recovery by following equation.
Recovery (%) = ([mixed sample] - [base sample]) / [8-OHdG]

Recovery (%) is expected to be between 80% and 120%.
Trouble shooting
1) If the result of recovery test is out of 80% - 120%, please dilute the filtrate using PBS (pH7.4) by 2-4 times.
2) If the result of ELISA is not stable, please measure in triplicate (N=3). Please refer technical information of 8-OHdG ELISA.
Assay example for human serum 8-OHdG
0.1 - 0.3 ng/mL in healthy person, and 0.7 ng/mL in patients with kidney diseases.
In some cases, the results would be below detection limit ( < 0.125 ng/mL).
References
1) Suzuki K, et. al.: The relationship between smoking habits and serum levels of 8-OHdG, oxidized LDL antibodies, Mn-SOD and carotenoids in rural Japanese residents. J Epidemiol 13(1), p29-37 (2003)
2) Michael J Forlenza and Gregory E Miller: Increased serum levels of 8-hydroxy-2'-deoxyguanosine in clinical depression. Psychosomat Med 68, p1-7 (2006)
Highly Sensitive 8-OHdG Check ELISA Technical Information
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