Download biomedica product listOxyStat Assay | BI-5007
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Method
Colorimetric assay, 96 wells
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Sample type
Serum, EDTA plasma, biological fluids (cell culture supernatant after matrix check)
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Sample volume
10 µl / well
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Assay time
15 min
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Sensitivity
7 µmol/l
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Standard range
0 – 600 µmol/l
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Use
Research use only
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Validation Data
See validation data tap for: precision data and values of healthy donors.
OxyStat Assay Product Overview
Colorimetric test system for the determination of the total oxidative status/capacity (TOS/TOC) in EDTA plasma, serum and other biological samples.
The Biomedica OxyStat assay measures the total peroxide concentration of a sample, utilizing a quick and simple assay procedure. Results show a direct correlation between free radicals and circulating biological peroxides and thus allow the characterization of the oxidative status in biological samples.
OxyStat Assay Principle
The peroxide concentration is determined by reaction of the biological peroxides with peroxidase and a subsequent color-reaction using TMB as substrate. After addition of a stop solution, the colored liquid is measured photometrically at 450 nm. A calibrator is used to calculate the concentration of circulating biological peroxides in the sample (one point calibration).
OxyStat Kit Components
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Contents |
Description |
Quantity |
|
PLATE |
Microtiter strips in strip holder |
12 x 8 tests |
|
SOLA |
Solution A, sample buffer, ready to use |
1 x 25 ml |
|
SOLB |
Solution B, reaction buffer, ready to use |
1 x 1 ml |
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SOLC |
Solution C, enzyme solution, ready to use |
1 x 50 µl |
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SOLD |
Solution D, reconstitution solution, ready to use |
1 x 5 ml |
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CTRL 1+2 |
Controls, lyophilized. The concentration after reconstitution is stated on the label. |
3 sets, lyophilized |
|
CAL |
Calibrators, lyophilized. The concentration after reconstitution is stated on the label. |
3 vials lyophilized |
|
STOP |
Stop solution, sulphuric acid, ready to use |
1 x 6 ml |
Storage instructions: All reagents as supplied in the OxyStat assay are stable at 4°C until the expiry date stated on the label of each reagent.
Sample Collection & Storage
Serum, EDTA plasma, cell culture supernatant (after matrix check) and other biological fluids are suitable for use in this oxidative stress assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, controls and the calibrator. The sample collection and storage conditions listed are intended as general guidelines.
EDTA Plasma and Serum
We recommend EDTA plasma as sample type since a time dependent increase in peroxide concentration is observed in serum samples. When serum is chosen, please make sure that during preparation of serum a time period no longer than 30 min at room temperature is allowed for clotting. Whole blood cannot be used in this assay. Heparın plasma, lipemic or heaemolytic samples may give erroneous results.
Collect venous fasting blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA as an anticoagulant. Allow Serum samples to clot for a maximum of 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -20°C.
Cell Culture Supernatant
In general, it is possible to determine the concentration of peroxides in cell culture supernatants when there is no matrix interference of the cell culture medium. This can be tested as follows:
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1. |
Dilute 10 µl of 30% H2O2 concentrate with 1000 µl of sample buffer A (SOLA) = S0. |
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2. |
Dilute 5 µl of S0 with 1000 µl of sample buffer A (SOLA) = S1 |
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3. |
Preparation 1: Pipet 10 µl cell culture medium into one well. |
|
4. |
Preparation 2: Pipet 10 µl aqua bidest. into another well. |
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5. |
Add each 10 µl S1 to both preparations. |
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6. |
Add each 100 µl sample buffer A (SOLA) and 100 µl reaction buffer mixture (ABC reaction-mix, see reagent preparation). |
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7. |
Incubate for 5 min. Add 50 µl stop solution (STOP). |
|
8. |
Measure immediately at 450 nm in a microtiter plate reader. |
Evaluation of the cell culture medium results:
A ratio of ODpreparation1:ODpreparation2 > 0.8 demonstrates that there are no major disturbing factors in the tested cell medium, and the assay can be performed.
Reagent Preparation
Calibrator & Controls
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1. |
Dissolve one CAL (calibrator) and one set of CTRL 1+2 (control) in 250 µl SOLD (reconstitution solution) each . The exact concentration is printed on the label of each vial. |
|
2. |
Leave at room temperature (18-26°C) for 5 min. Vortex gently. |
Reconstituted CAL and CTRLs can be stored aliquoted and frozen at -20°C up to one month. Avoid repeated freeze-thaw cycles.
ABC-Reaction-Mix
The following volume of ABC-reaction-mix is sufficient for 40 tests (wells) and should be used as guidance. However, the volume should be adjusted to the respective number of samples. Prepare the ABC-reaction-mix just before use in the assay by mixing:
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5 ml SOLA (sample buffer) + 100 µl SOLB (reaction buffer) + 5 µl SOLC (enzyme solution). |
Only use freshly prepared ABC-reaction-mix in the assay. Discard unused ABC-reaction-mix. Do not store for later use.
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.
Cloudy samples should be centrifuged for at least 5 min at 10 000xg. The supernatant is used in the test.
OxyStat Assay Protocol
Read the entire protocol before beginning the assay.
|
1. |
Bring samples and reagents to room temperature (18-26°C). |
|
2. |
Mark positions for CTRL/CAL/SAMPLE (control/calibrator/sample) on the protocol sheet. |
|
3. |
Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label. |
|
4. |
Add 10 µl CTRL/CAL/SAMPLE (control/calibrator/sample) into respective well. |
|
5. |
Add 100 µl SOLA (solution A, sample buffer) into each well. |
|
6. |
Measurement 1: determine absorption with a microplate reader at 450 nm. |
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7. |
Add 100 µl of the freshly prepared ABC-reaction-mix into all wells (see section Reagent Preparation). |
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8. |
Incubate 15 minutes at 37°C. |
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9. |
Add 50 µl STOP (stop solution) into each well. |
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10. |
Measurement 2: determine absorption with a microplate reader at 450 nm. |
Assay Procedure for Automated Pipetting Systems
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1. |
Adjust volumes of the reaction solutions and samples according to the analyser used. |
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2. |
If the analyser is incapable of adding the stop solution (STOP), perform kinetic measurements at 405 nm or at 580-620 nm without stopping the reaction. |
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3. |
Measurement time: 15 minutes at 20°C-37°C. |
Calculation of Results
The differences between measurement 1 and 2 are proportional to the peroxide concentrations of the samples.
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1. |
For each CTRL/CAL/SAMPLE (control/calibrator/sample) subtract the OD values of measurement 1 from the OD values of measurement 2 (= Δ OD). |
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2. |
A single-point calibration is done using the CAL (calibrator). The OD value is proportional to its concentration, which is stated on the label. This concentration is stated as H2O2 equivalents (µmol/l). |
|
3. |
The concentrations of controls and samples are calculated according to following formula: [µmol/l] sample = (Δ OD sample x [µmol/l] calibrator)/ (Δ OD calibrator) |
Background Information
Cells and tissues are sensitive to oxidative stress, caused by the formation of free radicals. If not deactivated by antioxidants, organic peroxides and hydroperoxides are the first reaction products between cellular constituents and free radicals or other reactive oxygen derivates.
The determination of the oxidative status / oxidative stress is essential in today's medical research and diagnostics. Methods used so far were either expensive (HPLC), or detected only degradation products of polyunsaturated fatty acids, like TBARS (thiobarbituric acid reactive substances).
The Biomedica OxyStat assay measures the total peroxide concentration of a sample, utilizing a quick and simple assay procedure. Results show a direct correlation between free radicals and circulating biological peroxides and thus allow the characterization of the oxidative status in biological samples.
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Cardiovascular Disease
Atherosclerosis
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Inflammation
Inflammatory processes
Sepsis
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Neurological Disorders
Neurdegenerative processes
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Oncology
Carcinogenesis
OxyStat Assay Precision
The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators using different ELISA lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run (intra-assay) precision was assessed by measuring 1 sample of known concentrations 12 times within one assay kit lot by one operator.
|
ID |
n |
Mean [µmol/l] |
SD [µmol/l] |
CV (%) |
|
Sample 1 |
12 |
221 |
6.9 |
3.1 |
In-Between-Run Precision
In-between-run (intra-assay) precision was assessed by measuring one sample twelve times within three assay kit lots by two different operators.
|
ID |
n |
Mean [µmol/l] |
SD [µmol/l] |
CV (%) |
|
Sample 1 |
12 |
221 |
11.9 |
5.1 |
OxyStat Assay Sample Values
Reference values from apparently healthy persons with no documented disease and medication:
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EDTA plasma [µmol/l] |
Serum [µmol/l] |
|
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Low oxidative stress |
<200 |
<180 |
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Moderate oxidative stress |
200-350 |
180-310 |
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High oxidative stress |
>350 |
>310 |
It is recommended to establish the normal range for each laboratory.
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