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NWLSSTM
Peroxiredoxin 3
ELISA
Product NWK-PRX03
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
nwkprx03rev031109

Simple ELISA kit for quantification of human
Peroxiredoxin 3 (Prx3) in biological samples.

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Table of Contents

SectionPageIntroduction

3

Intended Use3

Test Principle

3Specifications

4Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

5Warnings, 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

9

Performance Details

10

References

10

Statement of Limited Warranty

10

Notes

11

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Page 11

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:
Specificity
The following substances were tested and found to have no crossreactivity: human Prx1, Prx2, prx4, Prx5, Prx6, mouse Prx3 and rat Prx3.

Sensitivity
The minimal detectable dose of human Prx3 was calculated to be 2ng/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 = 5.0%
Inter-assay = 2.93%
Accuracy:
Recovery on addition from 92.9-103.6%,
Average = 98.2%
Recovery on dilution from 85-108.1, Average = 98.1%
Overall mean recovery = 98.15%
References
1. Sue Goo Rhee et al. (2005) Free Radic Biol Med.
38 (12):1543-52.
2. Fujii J, Ikeda Y. (2002) Redox Rep. 7 (3):123-30.
3. Sue Goo Rhee et al. (2001) IUBMB Life 52 (1-2):35-41.
4. Tong-Shin Chang et al. (2004) J Biol Chem. 279 (40):41975-84.
5. Larisa Nonn et al. (2003) Mol Cancer Res. 1 (9):682-9.
6. Zachary A. Wood et al. (2003) TIBS 28 (1): 32-40.

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Page 3Introduction:
Organisms living under aerobic conditions have developed various antioxidative mechanisms to protect them from damage by reactive oxygenspecies (ROS). Peroxiredoxin (Prx) is a growing anti-oxidative protein family
that has been identified six members in mammals. They share a common
reactive Cys residue in the N-terminal region, and are capable of serving asa peroxidase and involve thioredoxin and/or glutathione as the electron
donor. Prx1 to Prx4 have an additional Cys residue in the conserved Cterminal region, and are cross members as judged by the amino acidsequence similarity. Prx5 also contains an additional Cys in its C-terminal
region that is less conserved. On the other hand, Prx6 has only one uniqueCys. These Prx family members are distributed in subcellular localization,Prx1, 2, and 6 in cytosol, Prx3 in mitochondria, Prx4 in ER and secretion,Prx5 showing complicated distribution including peroxisome, mitochondria
and cytosol, all of which are potential sites of ROS production. In addition totheir role as a peroxidase, however, a body of evidence has accumulated tosuggest that individual members also serve divergent functions, which areassociated with various biological processes such as the detoxification ofoxidants, cell proliferation, differentiation and gene expression. It would beexpected that these functions might not necessarily depend on peroxidase
activity and, therefore, it seems likely that the divergence is due to uniquemolecular characteristics intrinsic to each member. The specific localizationof Prx3 in mitochondria together with the identification of its
mitochondria-specific electron suppliers, namely thioredoxin 2 andthioredoxin reductase 2, suggest that these three proteins might provide a
primary line of defense against H2O2 produced by the mitochondrial
respiratory chain. Furthermore, Prx3 expression is induced by oxidants inthe cardiovascular system and is thought to play a role in the antioxidant
defense system and homeostasis within mitochondria.
Intended Use:
The NWLSS™ Peroxiredoxin 3 (Prx3) ELISA kit is to be used for the invitro quantitative determination of human Prx3 in serum, plasma, cell lysate
and other biological samples. The assay will recognize both native andrecombinant human Prx3.
Test Principle:
The NWLSS™ Peroxiredoxin 3 (Prx3) assay is based on a sandwich EnzymeLinked Immunosorbent Assay (ELISA). The microtiter plate provided in thiskit has been pre-coated with a monoclonal antibody specific to human Prx3.
This stationary phase antibody binds sample or standard Prx3 while nonbound proteins are removed by washing. Next, bound Prx3 is tagged with abiotin-conjugated monoclonal antibody specific for Prx3 followed by Avidin
conjugated to Horseradish Peroxidase (HRP). Subsequent addition of TMBsubstrate solution causes blue color (650 nm) development proportional tothe amount of Prx3 originally captured by the stationary phase antibody.
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Specifications:
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1 X 96 well ELISA presented as 6 X 16 well (2 X 8 well)
Strips in frame.

Number of tests: Triplicate =Duplicate =2440Specificity:

Human Peroxiredoxin 3

Sensitivity:

2 ng/mL

Range:

2 ng/mL—32ng/mL

Kit Contents:
1 Foil Pouch

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Assay Protocol: (continued):
16. After appropriate incubation time, add 100ul 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.
Data Analysis:
1. Plot the mean absorbance at 450 nm for each standard versus thePeroxiredoxin 3 concentration. Select the best possible fit for the curve
obtained (4–parameter is recommended). This can typically be doneusing the software provided with most plate readers. An example curve isshown below.

96 well microplate precoated with anti-hu
Peroxiredoxin 3.

2. Sample Peroxiredoxin 3 is determined by comparing their absorbancemeasurements at 450 with those of the standard curve.

1 vialrHu-Peroxiredoxin 3 Standard (lyophilized)

1 bottle

Sample Dilution Buffer(25mL)

3. Sample data as read from the standard curve must be multiplied by thedilution factor used.

1 vial100X Secondary Antibody(Biotin labeled anti-hu Prx1)(150 uL)

1 bottle

Reagent Dilution Buffer(25mL)

1 vial100X Avidin-HRP Conjugate(150 uL)

1 bottle

Assay Preparation Buffer(30 mL)

3

1 bottle

TMB Substrate(20 mL)

2.5

1 bottle

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

1 bottle

10X Concentrated Wash Buffer(100 mL)

3

Adhesive Plate Covers(3)

Serological pipettes.
Deionized water.
Automatic plate washer or other aspiration devices are optional.

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Peroxiredoxin 3
Exam ple Standard Curve

Absorbance at 450 nmRequired Materials Not Provided:
Adjustable micropipettes with disposable tips (5-1000 L). Multi-channel
pipettes are useful and help to reduce intra-sample variability.

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.

21.5
1R2 = 10.5
00

1020

30

40

Peroxiredoxin 3 Concentration (ng/m L)
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Page 5Assay Protocol:
1. Add 300ul 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 uL 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 100ul of Diluted Standards to the appropriate microtiter wells and100ul of Sample Dilution Buffer to zero wells.
5. Add 100ul 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 100ul 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 100ul Working Conjugate Solution to each well.
12. Cover the plate with the plate cover and incubate for 30 minutes atroom temperature (20-25 °C).
13. Thoroughly aspirate or decant the solution from the wells. Wash thewells 3 times previously described in step 3.
14. Add 100ul of TMB Substrate to each well. The liquid in the wells

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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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.
All reagents containing Sodium Azide also contain Thimerosal as apreservative. Thimerosal contains Hg thus should be handled with greatcare.
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.
3. Bring all samples and reagents to room temperature before use.
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.
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. Equilibrate to room temperature, mix gently.
2. Mix 20ul of 100X Secondary Antibody with 2ml Reagent Dilution Bufferfor each 16 well strip to be assayed. Label as “Working Secondary Antibody Solution”.
3. Return the unused 100X Secondary Antibody to the refrigerator.
AVIDIN-HRP Conjugate
1. Equilibrate to room temperature, mix gently.
2. Mix 20ul 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.
Stop Solution
The Stop Solution is provided ready to use

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Page 7

Sample Handling/PreparationThe rate of degradation of native human Prx3 in various matrices has notbeen properly investigated. Therefore, it is beyond the scope of this publication to comment on specific sample processing protocols or necessary
sample dilutional schemes however here are some basic guidelines:
Plasma:Average normal peroxiredoxin 3 levels have been reported in the range of18 ng/mL. Therefore, a starting dilution of 2X to 5X is recommended forplasma from healthy (normal donors). Samples from oxidative stress or
known inflammatory model systems would be expected may contain significantly elevated levels of Prx3 such that higher dilutions may be necessary in some situations.
Tissue:Thioredoxin levels are expected to vary greatly in various tissue types
such that proper dilutional schemes for tissue homogenates must be
experimentally determined by the end user.
Standard Curve Preparation:
Reconstitute the human Prx3 standard to 1ug/ml by adding 1ml ofSample Dilution Buffer into the glass vial containing lyophilized humanPrx1 protein. Swirl or mix gently, and allow to sit for 5 minutes to ensurecomplete reconstitution.
1. Label tubes 1-8 tubes as:
32, 16, 8, 3.4, 2, 1 0.5 and zero (0) ng/mL.
2. Add 968 uL Standard Dilution Buffer to tube 1 and 500 uL Standard
Dilution Buffer to each tube 2-8.
3. Add 25 uL Reconstituted 1 ug/mL Standard to tube 1 and mix well.
4. perform a serial dilution by transferring 500 uL of 32 ng/mL Standard
into tube 2 mixing thoroughly then 500 uL of resulting 16 ng/mL to tube
3 and so on through tube 7 to create all standards down to 0.5 ng/mL.

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