Urinary 8-OHdG is a biomarker for oxidative damage, cancers, diabetes and
heart disease. The Assay Genie 8-OHdG ELISA Kit has been validated in urine and comes with
extensive validation data including Spike-recovery, Range and Linearity.
Furthermore the 8-OHdG ELISA is one of the most sensiteve kits on the market
to date, allowing researchers to forgo the laborious separation and isolation
steps of HPLC and detect low levels of 8-OHdG across any species.
Matrices listed below were spiked with certain level of 8-OHdG and the
recovery rates were calculated by comparing the measured value to the
expected amount of 8-OHdG in samples.
Matrix
Recovery range(%)
Average(%)
serum(n=5)
86-96
91
EDTA plasma(n=5)
86-101
93
UFH plasma(n=5)
85-105
96
Linearity:
The linearity of the kit was assayed by testing samples spiked with
appropriate concentration of 8-OHdG and their serial dilutions. The
results were demonstrated by the percentage of calculated
concentration to the expected.
Sample
1:2
1:4
1:8
serum(n=5)
86-104%
85-104%
87-104%
EDTA plasma(n=5)
86-100%
87-97%
90-99%
UFH plasma(n=5)
83-93%
84-100%
83-98%
Intra-Assay:
CV <8%
Inter-Assay:
CV <10%
Step
Procedure
1.
Set standard, test sample and control (zero) wells on the pre-coated
plate respectively, and then, record their positions. It is
recommended to measure each standard and sample in duplicate. Wash
plate 2 times before adding standard, sample and control (zero) wells!
2.
Add Sample and Biotin-detection antibody: Add 50µL of Standard,
Blank or Sample per well. The blank well is added with Sample Dilution
Buffer. Immediately add 50 µL of biotin-labelled antibody
working solution to each well. Cover with the plate sealer provided.
Gently tap the plate to ensure thorough mixing. Incubate for 45
minutes at 37°C. (Solutions are added to the bottom of micro-ELISA
plate well, avoid touching plate walls and foaming).
3.
Wash: Aspirate each well and wash, repeating the process three times.
Wash by filling each well with Wash Buffer (approximately 350µL)
using a squirt bottle, multi-channel pipette, manifold dispenser or
automated washer. Complete removal of liquid at each step is essential
to good performance. After the last wash, remove any remaining Wash
Buffer by aspirating or decanting. Invert the plate and pat it against
thick clean absorbent paper.
4.
HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working
solution to each well. Cover with a new Plate sealer. Incubate for 30
minutes at 37°C.
5.
Wash: Repeat the aspiration/wash process for five times.
6.
TMB Substrate: Add 90µL of TMB Substrate to each well. Cover
with a new plate sealer. Incubate for about 10-20 minutes at 37°C.
Protect from light. The reaction time can be shortened or extended
according to the actual color change, but not more than 30 minutes.
When apparent gradient appeared in standard wells, you can terminate
the reaction.
7.
Stop: Add 50µL of Stop Solution to each well. Color turn to
yellow immediately. The adding order of stop solution should be as the
same as the substrate solution.
8.
OD Measurement: Determine the optical density (OD Value) of each well
at once, using a microplate reader set to 450 nm. You should open the
microplate reader ahead, preheat the instrument, and set the testing
parameters.
When carrying out an ELISA assay it is important to prepare your samples
in order to achieve the best possible results. Below we have a list of
procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum:
If using serum separator tubes, allow samples to clot for 30 minutes
at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect
the serum fraction and assay promptly or aliquot and store the
samples at -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to
clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g.
Removeserum and assay promptly or aliquot and store the samples
at-80°C. Avoid multiple freeze-thaw cycles.
Plasma:
Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge
samples at 4°C for 15 mins at 1000 - g within 30 mins of
collection. Collect the plasma fraction and assay promptly or aliquot
and store the samples at -80°C. Avoid multiple freeze-thaw
cycles.Note: Over haemolysed samples are not
suitable for use with this kit.
Urine & Cerebrospinal Fluid:
Collect the urine (mid-stream) in a sterile container, centrifuge for
20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If
any precipitation is detected, repeat the centrifugation step. A
similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant:
Collect the cell culture media by pipette, followed by centrifugation
at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and
assay immediately.
Cell Lysates:
Solubilize cells in lysis buffer and allow to sit on ice for 30
minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove
insoluble material. Aliquot the supernatant into a new tube and
discard the remaining whole cell extract. Quantify total protein
concentration using a total protein assay. Assay immediately or
aliquot and store at ≤ -20°C.
Tissue Homogenates:
The preparation of tissue homogenates will vary depending upon tissue
type. Rinse tissue with 1X PBS to remove excess blood &
homogenizein 20ml of 1X PBS (including protease inhibitors) and store
overnight at ≤ -20°C. Two freeze-thaw cycles are required to
break the cell membranes. To further disrupt the cell membranes you
can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg.
Remove the supernatant and assay immediately or aliquot and store at
-20°C or-80°C.
Tissue Lysates:
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a
tissue homogenizer in PBS. Add an equal volume of RIPA buffer
containing protease inhibitors and lyse tissues at room temperature
for 30 minutes with gentle agitation. Centrifuge to remove debris.
Quantify total protein concentration using a total protein assay.
Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:
Collect milk samples and centrifuge at 10,000 x g for 60 min at
4°C. Aliquot the supernatant and assay. For long term use, store
samples at -80°C. Minimize freeze/thaw cycles.
