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P r e m ie r P r o d u c ts f o r S u p e r io r Lif e S c ie n c e R e s e a r c h

NWLSSTM
MnSOD ELISA
Product NWK-SOD04
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 ManganeseSuperoxide Dismutase (SOD2 or MnSOD) in biologicalsamples.

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Table of Contents

SectionPageIntroduction

3

Intended Use

3

Test Principle

3Specifications

4

Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

5

Warnings, Precautions, Limitations

5Storage 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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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 Cu/ZnSOD (SOD1), ecSOD (SOD3), SOD4 (CCS or copper chaperone), rat SOD2 and mouse SOD2.
Sensitivity
The minimal detectable dose of human MnSOD was calculated to be 25
pg/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 = 5.35 %Inter-assay = 6.28 %Accuracy:
Recovery on addition is 98.6~99.6%
Recovery on dilution is 98.8~104.5%(Average 99.1%)(Average 100.9%)

Overall mean recovery = 100%
References
1. Edward Luk et al manganese Activation of Superoxide Dismutase 2 in
the Mitochondria of Saccharomyces cerevisiae. (2005) The Jounal of Biol.
Chem. 280(24):22715-22720
2. Macmillan-Crow LA et al Invited review: manganese superoxide
dismutase in disease (2001) Free radic Res 34(4):325-336
3. Igor N. Zelko et al Superoxide dismutase multigene family: acomparison of the CuZn-SOD (SOD1), Mn-SOD(SOD2), and EC-SOD(SOD3)
gene structures, evolution, and expression (2002) Free Radical Biology &Medicine 33(3):337-349
4. Ruby Leah B. et al The copper Chaperone CCS Directly Interacts withCopper/Zinc Superoxide Dismutase (1998) The Journal of Biological
Chemistry 273(37):23625-23628
5. Tim D. Oury et al Human extracellular superoxide dismutase is atetramer composed of two disulphide-linked dimmers: a simplified, highyield purification of extracellular superoxide dismutase (1996
Biochem .J .317:51-57

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Page 3Introduction:
Superoxide dismutase (SOD) is an antioxidant enzyme involved in defense
against reactive oxygen species (ROS). SOD catalyzes the dismutation ofsuperoxide radical anion (O2-) to hydrogen peroxide, which is thencatalyzed to O2 and H2O by glutathione peroxidase and/or catalase.
Several classes of SOD have been discovered and can be differentiatedaccording to the metal associated with their active site as well as theirlocation within a cell or tissue. In mammals, Cu/ZnSOD (SOD1) islocalized in the cytosol and ecSOD (SOD3) in the extracellular matrix ofvarious tissues. MnSOD or SOD2 exists as a tetramer with individual
subunit MW of approximately 23 kDa and Mn at its active site. It is
initially synthesized with a leader peptide which targets this enzyme
exclusively to the mitochondrial spaces. MnSOD, as the primary
antioxidant enzyme for scavenging superoxide radicals in mitochondria, isessential for the survival of all aerobic organisms. Dysregulation ofMnSOD in the form of under-expression is being investigated relative tovarious disease states as well as aging. Overexpression of MnSOD has
been shown to protect against oxidative stress induced cell death andtissue injury. MnSOD has also been shown to play a major role inpromoting cellular differentiation and tumorigenesis and in protecting
against hyperoxia-induced pulmonary toxicity.
Intended Use:
The NWLSS™ MnSOD ELISA kit is intended to be used for the in vitro
quantitative determination of human MnSOD in cell lysate and tissue
homogenates. The assay will recognize both native and recombinanthuman MnSOD.
Test Principle:
The NWLSS™ MnSOD 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 humanMnSOD. This stationary phase antibody binds sample or standard
MnSOD while nonbound proteins are removed by washing. Next, bound
MnSOD is tagged with a biotin-conjugated monoclonal antibody specificfor MnSOD followed by Avidin conjugated to Horseradish Peroxidase(HRP). Subsequent addition of TMB-substrate solution causes blue color(650 nm) development proportional to the amount of MnSOD originallycaptured by the stationary phase antibody. Finally, addition of a sulfuric
acid solution stops the reaction resulting in a yellow color productmeasured at 450 nm. Sample MnSOD concentration is determined bycomparing the 450 nm absorbance of sample wells to the absorbance ofknown standards.

6. Richard W. Strange et al The Structure of Holo and Metal-dificient WildNwktsod04rev063009

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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 =2440

Human MnSOD

Sensitivity:

25 pg/mL

Range:

25 pg/mL—1600 pg/mL

Kit Contents:
1 Foil Pouch

96 well microplate precoated with anti-hu MnSOD.

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

Sample/Standard Dilution Buffer(25mL)

1 vial100X Secondary Antibody (Lyophiloized)(biotin labeled anti-hu MnSOD)(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 theMnSOD 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 MnSOD 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 thedilution 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.
MnSOD
Exam ple Standard Curve
1.61.4Absorbance at 450 nm

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1.2
10.8
0.6
R2 = 0.9998

0.4
0.2
00500

1000

1500

2000

Mn SOD Concentration (pg/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 optionalmonitoring 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 and100l 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.

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.

11. Add 100l Working Conjugate Solution to each well.

Assay Preparation:
1. Determine the number of wells required to assay standards, samples
and controls for the appropriate number of replicates. It is recommendedthat testing be performed in duplicate or triplicate if possible.

12. Cover the plate with the plate cover and incubate for 30 minutes atroom temperature (20-25 °C).

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 at2-8 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 ConjugateSolution”.
3. Return the unused 100X AVIDIN-HRP Conjugate to the refrigerator.

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Sample Handling/Preparation
The rate of degradation of native human MnSOD in various matrices has
not been investigated. It is beyond the scope of this publication to comment on specific sample processing protocols except to recommend thattissue or cell samples (eg samples where mitochondria and thus MnSOD
would be present) be homogenized in normal phosphate buffered saline
and treated as necessary (sonication) to appropriately disrupt the mitochondria in order to release MnSOD into the homogenate. Assuming a
relatively short half life for MnSOD outside of the mitochondria, homogenates should be kept as cold as possible and tested as soon as possible
after disruption of the mitochondrial membrane.
Dilutional Scheme:
MnSOD levels are expected to vary greatly in various tissue types suchthat proper dilutional schemes for tissue homogenates must be experimentally determined by the end user. We recommend starting with ahomogenate that is as concentrated as possible then making dilutions
such as 1:1, 1:4, 1: 9 as necessary to see at what point the best data isgenerated for a given sample type or model system.
Standard Curve Preparation:
Reconstitute the human MnSOD standard to 10ng/ml by adding 1ml ofSample/Standard Dilution Buffer into the standard protein glass vial
containing lyophilized human MnSOD protein. Swirl or mix gently, andallow to sit for 5 minutes to ensure complete reconstitution.

Wash Buffer
1. Equilibrate to room temperature, mix to re-dissolve any precipitatedsalt.

1. Label tubes 1-8 tubes as:
1600, 800, 400, 200, 100, 50, 25 and zero (0) pg/mL.

2. Mix 1 volume 10X Wash Buffer with 9 volumes of deionized water.
Label as “Working Wash Solution”.

2. Add 840 L Standard Dilution Buffer to tube 1 and 500 L StandardDilution Buffer to each tube 2-8.

3. Store both the remaining concentrated Wash Buffer and the Working
Wash Solution at 4°C in the refrigerator.

3. Add 160 L Reconstituted 10 ng/mL Standard to tube 1 and mix well.

TMB Substrate
The TMB Substrate is provided ready to use.

4. Make a serial dilution by transferring 500 L of 1600 pg/mL Standard
into tube 2 mixing thoroughly then 500 L of resulting 800 pg/mL totubes 3 and so on to create all Standards down to 25 pg/mL.

Stop Solution
The Stop Solution is provided ready to use

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