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NWLSSTM
Human
cGPx (GPX1) ELISA
Product NWK-GPX02
For Research Use Only

5131 NE 94th Avenue, Suite 201
Vancouver, WA 98662
Phone 360-449-3091 or Toll Free: 888-449-3091
Fax 360-449-3092
E-mail: sales@nwlifescience.com
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Simple ELISA kit for quantification of human cellular
Glutathione Peroxidase (GPX1) in biological samples.

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To Order Call: 1-888-449-3091

Page

Introduction

3

Intended Use

3

Test Principle

3

Specifications

4

Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

5

Warnings, Precautions, Limitations

5

Storage Instructions

5

Assay Preparation

5

Reagent Preparation

6

Sample Handling/Preparation

7

Standard Curve Preparation

7

Assay Protocol

8

Data Analysis

10

Performance Details

11

References

11

Statement of Limited Warranty

12

Notes

12

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References (cont.):7) Stefan Blankenberg, et al. Glutathione peroxidase 1 activity andcardiovascular events in patients with coronary artery disease (2003) N Eng
J Med 349:1605-1613

Table of Contents

SectionTo Order Call: 1-888-449-3091

Statement of Limited Warranty:
Northwest Life Science Specialties, LLC (NWLSS) makes no guarantee ofany kind, expressed or implied, that extends beyond the description of thematerial in this kit, except that they will meet our specifications at the timeof delivery. Customer's remedy and NWLSS’ sole liability is limited to, at
NWLSS’ option, refund of the purchase price, or the replacement ofmaterial not meeting our specification. By acceptance of our product, customer assumes all liability and will indemnify and hold NWLSS harmless forthe consequence of this product’s use or misuse by the customer, its
employees, or others. Refund or replacement is conditioned of customer
notifying NWLSS within twenty-one (21) days of the receipt of product.
Failure to give notice within 21 days shall constitute a waiver by the customer of all claims hereunder with respect to said product..
Notes:

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Performance Details:
The following substances were tested and found to have no crossreactivity: human GPx3, human GPx4, Rat GPx1 and mouse GPx1.
Sensitivity
The minimal detectable dose of human cGPx was calculated to be 1.56
ng/ml, by subtracting two standard deviations from the mean of 10 zerostandard replicates (ELISA buffer, S0) and intersecting this value with thestandard curve obtained in the same calculation.
Precision
Intra-assay = 3.65 %Inter-assay = 6.48%
Accuracy:
Recovery on addition is 99.3~101.2%
Recovery on dilution is 97.9~103.4%(Average 99.5%)(Average 100.7%)

Overall mean recovery = 102.3%
References:
1) Takebe, G.. et al. A comparative study on the hydroperoxide and thiol
specificity of the glutathione peroxidase family and selenoprotein P(2002) J. Biol. Chem. 277(43): 41254-41258
2) Avissar, N. et al. Human placenta makes extracellular glutathione
peroxidase and secretes it into maternal circulation (1994) Am. J. Physiol.
267: E68-76
3) Bao, Y. et al. Reduction of thymine hydroperoxide by phospholipidhydroperoxide glutathione peroxidase and glutathione transferases(1997) FEBS Lett. 410: 210-212
4) Chambers, I. et al. The structure of the mouse glutathione peroxidase
gene: the selenocysteine in the active site is encoded by the 'termination'codon, TGA (1986) EMBO J. 5: 1221-1227
5) Perry, A. et al. Genetic evidence for an androgen-regulated epididymal
secretory glutathione peroxidase whose transcript does not contain aselenocysteine codon (1992) Biochem. J. 285: 863- 870
6) De Haan J. et al. An imbalance in antioxidant defense affects cellularfunction: the pathophysiological consequences of a reduction in
antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout
mouse (2003) Redox Rep. 8(2): 69-79
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Page 3Introduction:
Glutathione Peroxidase (GPx) is a family of ubiquitously expressed proteins
which catalyze the reduction of hydrogen peroxides and organichydroperoxides by glutathione. Several isoforms exist that differ in theirprimary structure and location. The classical cytosolic/mitochondrial Gpx1
(cGpx) is selenium dependent and the first of the Gpx family to bediscovered. A gastrointestinal form (GiGpx or GPX2), is an intracellularenzyme expressed only in GI system epithelial cells. An extra-cellular form
(plGpx or GPx3) is mainly expressed by the kidney where it is released into
the blood circulation. Phospholipid hydroperoxide (PhGpx or GPx4)
expressed in most tissues, can reduce many hydroperoxides including those
integrated into membranes and hydroperoxy lipids in low density lipoprotein
or thymine. Mammalian GPx family members, except for the recently
described Cys containing GPx3 and epididymis-specific secretory GPx (eGpxor GPx5), possess selenocysteine at the active site. Because GPx1 plays amajor role in the prevention of oxidative stress, it may also be an importantanti-atherogenic enzyme. In mice, cGPx deficiency results in abnormal
vascular and cardiac function and structure. From a clinical perspective, it
has been reported that low erythrocyte GPx1 activity identifies patients withcoronary artery disease who are at the high risk for cardiovascular events.
The measurement of GPx1 activity therefore could provide additional
information on risk and thus might be another useful biomarker in identifying
patients who would benefit from preventive antioxidant treatment.
Intended Use:
The NWLSS™ cGPx ELISA kit is intended to be used for the in vitro
quantitative determination of human cGPx in cell lysate and tissue homogenates. The assay will recognize both native and recombinant human cGPx.
Test Principle:
The NWLSS™ cGPx Assay is based on a sandwich Enzyme-Linked
Immunosorbent Assay (ELISA). The microtiter plate provided in this kit has
been pre-coated with a monoclonal antibody specific to human cGPx. This
stationary phase antibody binds sample or standard cGPxwhile nonboundproteins are removed by washing. Next, bound cGPx is tagged with a biotin
conjugated monoclonal antibody specific for cGPx followed by Avidin
conjugated to Horseradish Peroxidase (HRP). Subsequent addition of TMB
substrate solution causes blue color (650 nm) development proportional tothe amount of cGPx originally captured by the stationary phase antibody.
Finally, addition of a sulfuric acid solution stops the reaction resulting in ayellow color product measured at 450 nm. Sample cGPx concentration isdetermined by comparing the 450 nm absorbance of sample wells to theabsorbance of known standards.

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Specifications:
Format::To Order Call: 1-888-449-3091

1 X 96 well ELISA presented as 6 X 16 well (2 X 8 well)
Strips in frame.

Number of tests: Triplicate =Duplicate =24
40

Human cGPx

Sensitivity:

12.5 pg/mL

Range:

12.5 pg/mL—800 pg/mL

Kit Contents:
1 Foil Pouch

96 well microplate precoated with anti-hu cGPx.

1 vialrHu-cGPx Standard (lyophilized)(1 Vial)1 Bottle

Sample/Standard Dilution Buffer(25mL)

1 vial100X Secondary Antibody (Lyophilized)(biotin labeled anti-hu cGPx)(1 Vial)1 Bottle

Reagent Dilution Buffer(25mL)

1 vial100X Avidin-HRP Conjugate(150 L)

1 Bottle

Assay Preparation Buffer(30 mL)

1 Bottle

TMB Substrate Solution(20 mL)

1 Bottle

Stop Solution (1 N Sulfuric Acid)(20 mL)

1 Bottle

10X Concentrated Wash Buffer(100 mL)

3

Adhesive Plate Covers(3)

Data Analysis:
1. Plot the mean absorbance at 450 nm for each standard versus thecGPx concentration. Select the best possible fit for the curve
obtained(4–parameter is recommended). This can typically be done using thesoftware provided with most plate readers. An example curve is shown
below.

Required Materials Not Provided:
Adjustable micropipettes with disposable tips (5-1000 L). Multi-channel
pipettes are useful and help to reduce intra-sample variability.

Deionized water.

Assay Protocol: (continued):
16. After appropriate incubation time, add 100l of Stop Solution to eachwell. The solution in the wells should change from blue to yellow.
17. Read and record the absorbance of each well at 450nm within 20
minutes of adding the Stop Solution.

Specificity:

Serological pipettes.

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2. Sample cGPx is determined by comparing their absorbancemeasurements at 450 with those of the standard curve.
3. Sample data as read from the standard curve must be multiplied by the
dilution factor used.
Note: Samples with an ABS450 exceeding that of the highest standard
should be additionally diluted with Sample Dilution Buffer and re-assayedin order to avoid erroneous results.
Hum an cGPx
Exam ple Standard Curve
3
Absorbance at 450 nm

Page 4

2.5
2
1.5
1
0.5
0
0

50

100

150

cGPx Concentration (ng/m L)

Automatic plate washer or other aspiration devices are optional.
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Assay Protocol:
1. Add 300l of Assay Prep Buffer to all wells and incubate the plate for 5
minutes at room temperature.

Required Instrumentation:
Plate reader with 450 nm capability (650 nm is required for optional
monitoring of color development prior to stopping the reaction).

2. Thoroughly aspirate or decant the solution from the wells.

Warnings, Precautions & Limitations:
Reagents are intended for research use only and are not for use indiagnostic or therapeutic procedures.

3. Wash wells 2 times as follows: Dispense 300 L Working Wash
Solution to each well and allow to soak for 1-3 minutes before decanting
or aspirating the remaining solution from the wells.
4. Add 100l of Diluted Standards to the appropriate microtiter wells and100ul of Sample Dilution Buffer to zero wells.
5. Add 100l of Sample to each well according to plan.
6. Cover the plate with the plate cover and incubate for 2 hours at room
temperature.
7. Aspirate or decant the solution from the wells then wash the wells 3
times as previously described in step 3.
8. Add 100l of Working Secondary Antibody to each well.
9. Cover the plate with the plate cover and incubate for 1 hour at room
temperature (20-25 °C).
10. Aspirate or decant the solution from the wells then wash the wells 3
times as previously described in step 3.
11. Add 100l Working Conjugate Solution to each well.
12. Cover the plate with the plate cover and incubate for 30 minutes atroom temperature (20-25 °C).

Individual components may be harmful if swallowed, inhaled or absorbed
through the skin. Contact should be minimized through the use of gloves
and standard good laboratory practices. If contact with skin or eyes
occurs, rinse the site immediately with water and consult a physician.
Substrate solutions must be at room temperature prior to use. Avoid
contact of substrate solutions with oxidizing agents and metal.
Improper or insufficient washing at any stage of the procedure will
result in either false positive or false negative results. Completely
empty wells before dispensing fresh Wash Buffer, fill with Wash
Buffer as indicated for each wash cycle and do not allow wells to sit
uncovered or dry for extended periods.
Storage Instructions:All kit components of this kit are stable at 2 to 8 ℃. Any unusedreconstituted standard should be discarded or frozen at -70 ℃. Standard
can be frozen and thawed one time only without loss of immunoreactivity.
Assay Preparation:
1. Determine the number of wells required to assay standards, samples
and controls for the appropriate number of replicates. It is recommended
that testing be performed in duplicate or triplicate if possible.
2. Create an assay template showing positioning of standards, controls
and samples.

13. Thoroughly aspirate or decant the solution from the wells. Wash thewells 3 times previously described in step 3.

3. Bring all samples and reagents to room temperature before use.

14. Add 100l of TMB Substrate to each well. The liquid in the wells

4. To avoid condensation, do not open foil pouches containing the microtiter strips until after they have reached room temperature. Next remove
the required number of strips and place in the frame supplied.

should begin to turn blue.

15. Incubate the plate at room temperature for approximately 10-15
minutes.
Note: The incubation time for the TMB substrate is dependent on ambientconditions as well as the specific microtiter plate reader in use. The user
should adjust this time as necessary by monitoring the development ofblue color at 650 nm and stopping when the high standard has reachedmaximal absorbance level.

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Return unused wells to the storage bag with desiccant, seal and store at
2-8 ºC.

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Reagent Preparation:
Assay Preparation Buffer
The Assay Preparation Buffer is provided ready to use.
Secondary Antibody
1. Reconstitute 100X Secondary Antibody by adding 150 L Reagent
Dilution Buffer to the vial.
2. Equilibrate 100X Secondary Antibody to room temperature, mix gently.
3. Mix 20l of 100X Secondary Antibody with 2ml Reagent Dilution Buffer
for each 16 well strip to be assayed. Label as “Working Secondary Antibody Solution”.
4. Return the unused 100X Secondary Antibody to the refrigerator.
AVIDIN-HRP Conjugate
1. Equilibrate to room temperature, mix gently.
2. Mix 20l of 100X AVIDIN-HRP Conjugate with 2ml Reagent Dilution
Buffer for each16-well strip to be assayed. Label as “Working Conjugate
Solution”.
3. Return the unused 100X AVIDIN-HRP Conjugate to the refrigerator.
Wash Buffer
1. Equilibrate to room temperature, mix to re-dissolve any precipitatedsalt.
2. Mix 1 volume 10X Wash Buffer with 9 volumes of deionized water.
Label as “Working Wash Solution”.
3. Store both the remaining concentrated Wash Buffer and the Working
Wash Solution at 4°C in the refrigerator.
TMB Substrate
The TMB Substrate is provided ready to use.

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Sample Handling/Preparation
The rate of degradation of native human cGPx (GPx1) in various matrices
has not been fully investigated. It is beyond the scope of this publication
to comment on specific sample processing protocols except to recommend that tissue or cell samples be homogenized in normal phosphatebuffered saline and treated as necessary to appropriately disrupt the cell
membranes in order to release cGPx into the homogenate. Assuming a
relatively short half life for cGPx outside of the cell, homogenates should
be kept as cold as possible and tested as soon as possible after creating
the lysate or homogenate.
Dilutional Scheme:
cGPx levels are expected to vary greatly in various tissue types such thatproper dilutional schemes for tissue homogenates must be experimentally determined by the end user. We recommend starting with a homogenate that is as concentrated as possible then making dilutions such as
1/2, 1/5, 1/10, etc. as necessary to see at what point the best data is
generated for a given sample type or model system.
Standard Curve Preparation:
Reconstitute the human cGPx standard to 1g/ml by adding 1ml ofStandard/Sample Dilution Buffer into the standard protein glass vial
containing lyophilized human cGPx protein. Swirl or mix gently, and allow
to sit for 5 minutes to ensure complete reconstitution.
1. Label tubes 1-8 tubes as:
100, 50, 25, 12.5, 6.25, 3.13, 1.56 and zero (0) ng/mL.
2. Add 900 L Standard Dilution Buffer to tube 1 and 500 L Standard
Dilution Buffer to each tube 2-8.
3. Add 100 L Reconstituted 1 g/mL Standard to tube 1 and mix well.
4. Make a serial dilution by transferring 500 L of 100 ng/mL Standard
into tube 2 mixing thoroughly then 500 L of resulting 50 ng/mL to tubes
3 and so on to create all Standards down to 1.56 ng/mL.

Stop Solution
The Stop Solution is provided ready to use

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