Human Superoxide Dismutase 1
ELISA
Catalog # LF-EK0101 (1 kit)
Catalog # LF-EK0102 (4 kits bundle)
Catalog # LF-EK0103 (10 kits bundle)

Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection ofhuman SOD 1 (Cu/Zn SOD)

For research use only
Not for diagnostic or therapeutic purposes1

Contents
1. Introduction ······························································

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2. Principles of Method ···················································

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3. Intended Use ·····························································

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4. Storage and Stability ···················································

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5. Chemical Hazard ·······················································

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6. Kit Contents ·····························································

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7. Materials Required But Not Provided ·····························

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8. Reagent preparation ···················································

61) Human SOD1 standard ···········································

62) Secondary Antibody ···············································

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3) AV-HRP ····························································

74) Washing buffer ····················································

75) Sample preparation···············································

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9. Assay Procedure ························································

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10. Characteristics ··························································

91) Typical result ······················································

92) Sensitivity ·························································

103) Specificity ·························································

104) Precision ··························································

115) Recovery ··························································

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11. Troubleshooting ························································

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12. Reference ································································

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1. Introduction
Superoxide dismutase (SOD) is an antioxidant enzyme involved in the defense systemagainst reactive oxygen species (ROS). SOD catalyzes the dismutation reaction of superoxide
radical anion (O2-) to hydrogen peroxide, which is then catalyzed to innocuous O2 and H2O byglutathione peroxidase and catalase. Several classes of SOD have been identified. : Cu/Zn SOD(SOD1) is localized in cytosol, Mn SOD (SOD2) in mitochondria, and ecSOD (SOD3) inextracellular space.
Cu/Zn SOD (SOD1) is a homodimer containing copper and zinc that is found almost
exclusively in intracellular cytoplasmic spaces.
The level of SOD1 elevates in response to a wide array of mechanical, chemical andbiological messengers such as heat shock, shear stress, and UVB- and X-irradiation. Decreased
levels of SOD1 expression can also be triggered by activation of the AP2 transcription factor. A
down-regulation of SOD1 has been shown in alveolar type II epithelial cells and lungfibroblasts after exposure to hypoxia.
The genomic organization of SOD1 genes shows striking similarities between species andconsists of five exons and four introns. The TATA and CCAAT boxes, as well as several highly
conserved GC-rich regions, have been localized in the proximal promoter region. Also, morethan 90 different mutations of the SOD1 gene have been associated with amyotrophic lateralsclerosis (ALS), also known as Lou Gehrig’s disease.

2. Principles of Method
The design of this 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 SOD1. Samples are pipetted into these wells. Nonbound SOD1 andother components of the sample should be removed by washing, then biotin-conjugatedmonoclonal antibody specific to SOD1 added. In order to quantitatively determine the amountof SOD1 present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) should be
added to each microplate well. The final step, a TMB-substrate solution added to each well.
Finally, a sulfuric acid solution is added and the resulting yellow colored product is measured at
450nm. Since the increases in absorbency is directly proportional to the amount of captured
SOD1.

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3. Intended Use
The AbFrontier human Superoxide Dismutase-1 (human SOD1) ELISA kit is to be used
for the in vitro quantitative determination of human SOD1 in human serum, human plasma,cell lysate and buffered solution. The assay will recognize both native and recombinanthuman SOD1.

This kit has been configured for research use only and is not to be used indiagnostic procedures.

4. Storage and Stability
All kit components of this kit are stable at 2 to 8
should be discarded or frozen at -70℃. Any unused reconstituted standard℃. Standard can be frozen and thawed one time only

without loss of immunoreactivity.

5. Chemical Hazard- Stop solution: This reagent is an irritant to eyes, skin and mucous membranes. Avoid
contact with eyes, skin and clothing. Wear suitable protective clothing, gloves and eye
protection. In the event of contact with eyes or skin, wash immediately with plenty ofwater.

- Standard protein and 2 Antibody containing Sodium Azide as a preservative.
nd4

6. Kit Contents
Contents

NumberVolume1 (in aluminum foil bag with desiccant)96 Well Plate
Incubation Buffer

1

30 ml

Washing Buffer2(20) 25 ml
1 Glass vial (lyophilized)

Standard Protein
1Standard/Sample Dilution Buffer

25 ml
1 Glass vial (lyophilized)

Secondary Antibody
1

150 µl1

25 ml

Substrate (TMB)

1

15 ml

Stop Solution

1

15 ml

AV-HRP
Secondary Antibody/AV-HRP
Dilution Buffer

Protocol booklet

1

Plate sealers

2① 96 Well Plate: Human SOD1 microtiter plate, one plate of 96 wells (8 well strips12).
A plate using break-apart strips coated with a mouse monoclonal antibody specific tohuman SOD1.

② Standard Protein: Recombinant human SOD1.

③ Secondary Antibody: Biotinylated anti human SOD1 antibody.

④ AV-HRP: Avidin linked Horseradish Peroxidase (HRP, enzyme)⑤ Substrate (Stabilized chromogen): Tetramethylbenzidine (TMB) solution⑥ Stop Solution: 1 N solution of sulfuric acid (H2SO4)⑦ Plate sealer: Adhesive sheet
 Do not mix or interchange different reagents from various kit lots.

57. Materials Required But Not Provided① Microtiter plate reader capable of measurement at or near 450nm.
② Calibrated, adjustable precision pipettes, preferably with disposable plastic tips (Amanifold multi-channel pipette is desirable for large assays.)③ Distilled or deionized water④ Data analysis and graphing software⑤ Vortex mixer⑥ Polypropylene tubes for diluting and aliquoting standard⑦ Absorbent paper towels⑧ Calibrated beakers and graduated cylinders of various sizes
8. Reagent Preparation
1) Human SOD1 standard

1. Reconstitute the lyophilized Human SOD1 standard by adding 1 ml ofStandard/Sample Dilution Buffer to make the 10 ng/ml standard stock solution. Allow
solution to sit at RT for 5 minutes, then gently vortex to mix completely. Use within one
hour of reconstituting.
2. Prepare 1 ml of 800 pg/ml top standard by adding 80 µl of the above stock solution in
920 µl of Standard/Sample Dilution Buffer. Perform 2-fold serial dilutions of the top
standards to make the standard curve within the range of this assay (12.5 pg/ml ~ 800
pg/ml) as below. Standard/Sample Dilution Buffer serves as the zero standard(0 pg/ml).
Standard

Add

Into

800.0 pg/ml

80.0 µl of the std.(10 ng/ml)

920.0 µl of the Standard/Sample Dilution Buffer

400.0 pg/ml

500 µl of the std.(800 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

200.0 pg/ml

500 µl of the std.(400 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

100.0 pg/ml

500 µl of the std.(200 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

50.0 pg/ml

500 µl of the std.(100 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

25.0 pg/ml

500 µl of the std.(50 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

12.5 pg/ml

500 µl of the std.(25 pg/ml)

500.0 µl of the Standard/Sample Dilution Buffer

0 pg/ml

1 ml of the Standard/Sample Dilution Buffer2) Secondary Antibody
100X secondary antibody solution can be made by adding 150 µl secondary antibody/AVHRP dilution buffer in the vial.
1. Equilibrate to room temperature, mix gently.

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2. Mix 20 µl Secondary Antibody concentrated solution (100X) + 1.98 ml Secondary
Antibody/AV-HRP dilution buffer. (Sufficient for two 8-well strip, prepare more if
necessary)

Label as “Working Secondary antibody Solution”.

3. Return the unused Secondary Antibody concentrated solution to the refrigerator.
3) AV-HRP
1. Equilibrate to room temperature, mix gently.
2. Mix 20 µl AV-HRP concentrated solution (100X) + 1.98 ml Secondary Antibody/AVHRP dilution buffer. (Sufficient for two 8-well strip, prepare more if needed)
Label as “Working AV-HRP Solution”.
3. Return the unused AV-HRP concentrated solution to the refrigerator.
4) Washing buffer
1. Equilibrate to room temperature, mix to re-dissolve any precipitated salt.
2. Mix 0.5 volume Wash buffer concentrate solution (20X) + 9.5 volumes of deionizedwater. Label as “Working Washing Solution”.
3. Store both the concentrated and the Working Washing Solution in the refrigerator.
* Directions for washing
1. Fill the wells with 300 µl of “Working Washing Buffer”.
Let soak for 1 to 3 minutes and then all residual wash-liquid must be drained from thewells by aspiration (taking care not to scratch the inside of the well) or decantation,
followed by forceful tapping of the plate on absorbent paper. Never insert absorbent
paper directly into the wells.
If using an automated washer, the operating instructions for washing equipment should
be carefully followed.
2. Incomplete washing will adversary affects the assay and renders false results.
3. It is recommended to use laboratory tape to hold plate strips to the plate frame while
performing the plate washing to avoid strips coming free of the frame.
5) Sample preparation
Blood should be collected by veinpuncture. For plasma samples, blood may be drawn into
tubes containing sodium citrate or heparin, EDTA. The serum or plasma should be separated
from the coagulated or packed cells by centrifugation. Specimens may be shipped at room
temperature and then stored refrigerated at 2-8℃ if testing is to take place within one week

after collection. If testing is to take place later than one week, specimens should be stored at 20℃. Avoid repeated freeze/thawing.

79. Assay Procedure Allow all reagents to reach room temperature before use. Gently mix all liquid reagentsprior to use.
 All standards, controls and samples should be run in duplicate for confirmation of
reproducibility.
 A standard curve must be run with each assay.
 If particulate matter is present in the analyte, centrifuge or filter prior to analysis.
 Maintain a consistent order of components and reagents addition from well to well.
This ensures equal incubation times for all wells.

1) Determine the number of 16-well strips needed for assay. Insert these in the flame(s) for
current use (Re-bag extra strips and frame. Refrigerate for further use).
2) Add 300 µl of Incubation buffer to all wells and incubate the plate for 5 minutes at room
temperature.
3) Thoroughly aspirate or decant the solution from the wells. Wash wells 2 times (See“Directions for washing”).
4) For the standard curve, add 100 µl of the standard to the appropriate microtiter wells.
Add 100 µl of the Standard/Sample Dilution Buffer to zero wells.
5) Serum and plasma require at least 20 fold dilution in the Standard/Sample Dilution
Buffer. And add 100 µl of samples to each wells.
6) Cover the plate with the plate cover and incubate for 2 hours at room temperature.
7) Thoroughly aspirate or decant the solution from the wells. Wash the wells 3 times (See“Directions for washing”).
8) Pipette 100 µl of “Working Secondary Antibody Solution” into each well.
9) Cover the plate with the plate cover and incubate for 1 hour at room temperature.
10) Thoroughly aspirate or decant the solution from the wells. Wash the wells 3 times (See“Directions for washing”).
11) Add 100 µl “Working AV-HRP Solution” to each well.
12) Cover the plate with the plate cover and incubate for 30 minutes at room temperature.
13) Thoroughly aspirate or decant the solution from the wells. Wash the wells 3 times (See“Directions for washing”).
14) Add 100 µl of Substrate to each well. The liquid in the wells should begin to turn blue.
15) Incubate the plate at room temperature.
 Do not cover the plate with aluminum foil, or color may develop.
The incubation time for chromogen substrate is often determined by the microtiter plate
reader used. O.D. values should be monitored and the substrate reaction stopped before
8O.D. of the positive wells exceeds the limits of the instrument. O.D. values at 450nm
can only be read after the Stop Solution has been added to each well.
 Because the Substrate is light sensitive, avoid the remained Substrate solution
prolonged exposure to light.
 Typically, reaction is stopped 5~10 minutes after treatment of Substrate, but this time
can be adjusted as the user desires.
16) Add 100 µl of Stop Solution to each well. The solution in the wells should change fromblue to yellow.
17) Read the absorbance of each well at 450 nm. Read the plate within 20 minutes of adding
the Stop Solution.
18) Plot on graph paper the absorbance of the standard against the standard concentration(Optimally, the background absorbance can be subtracted from all data points, including
standards, unknowns and controls, prior to plotting.). Draw a smooth curve through thesepoints to construct the standard curve.
19) Read the human SOD1 concentrations for the unknown samples and controls from thestandard curve plotted in step 18. Multiply value(s) obtained for the unknown sample by
the dilution factor (Samples producing signals greater than that of the highest standard
should be further diluted in the Standard/Sample Dilution Buffer).

10.Characteristics
1) Typical result
The standard curve below is for illustration only and should not be used to calculate
results in your assay.

A standard curve must be run with each assay.
Standard

Optical Density

Human SOD1 (pg/ml)(at 450nm)

0

0.093

12.5

0.102

25.0

0.126

50.0

0.179

100.0

0.289

200.0

0.502

400.0

0.880

800.0

1.754

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< Limitations > Do not extrapolate the standard curve beyond the 800 pg/ml standard point.
 Other buffers and matrices have not been investigated.
 The rate of degradation of native human SOD1 in various matrices has not been
investigated.

2) Sensitivity
The minimal detectable dose of human SOD1 was calculated to be 12.5 pg/ml, by
subtracting two standard deviations from the mean of 10 zero standard replicates (ELISA
buffer, S0) and intersecting this value with the standard curve obtained in the same
calculation.
3) Specificity
The following substances were tested and found to have no cross-reactivity: human SOD2,
SOD3, SOD4, rat SOD1 and mouse SOD1.

104) Precision① Within-Run (Intra-Assay)(n=5)
Mean (pg/ml)

SD (pg/ml)

CV (%)

204.164.23

2.1

400.7430.84

7.7

798.2451.20

6.4② Between-Run (Inter-Assay)(n=8)
Mean (pg/ml)

SD (pg/ml)

CV (%)

82.76

7.91

9.6

177.7912.35

6.9374.2918.92

5.15) Recovery
Recovery on addition is 99.4~99.5 % (mean 99.5 %)Recovery on dilution is 98.0~106.1 % (mean 100.7 %)11

11.Troubleshooting
ProblemPossible Cause• Insufficient washing

Solution• Increase number of washes• Increase time of soaking
between in wash

High signal and backgroundin all wells• Too much AV-HRP• Check dilution, titration• Incubation time too long

• Reduce incubation time

• Development time too long

• Decrease the incubation time
before the stop solution is
added• Reagent added in incorrect• Review protocol

order, or incorrectly preparedNo signal• Standard has gone bad• Check the condition of stored(If there is a signal in thestandard

sample wells)• Assay was conducted from • Reagents allows to come to
an incorrect starting point

20~30℃ before performing

assay• Insufficient washing• Increase number of washes– unbound AV-HRP remaining

carefully

Too much signal – whole• Too much AV-HRP• Check dilution

plate turned uniformly blue

• Plate sealer or reservoir• Use fresh plate sealer andreused, resulting in presence

reagent reservoir for eachof residual AV-HRP

step• Plate not developed long

• Increase substrate solution

Enoughincubation time

• Improper calculation of• Check dilution, make newstandard curve dilution

standard curve

No signal when a signal is• Sample matrix is masking• More diluted sample

expected, but standard curve

detection

Standard curve achieved but
poor discrimination between
point

recommended

looks fineSamples are reading too high,but standard curve is fine

Edge effect• Samples contain protein • Dilute samples and run
levels above assay range

again• Uneven temperature around• Avoid incubating plate inwork surface

areas where environmental
conditions vary
• Use plate sealer

1212.Reference
1) Ruby Leah B. et al. The copper Chaperone CCS Directly Interacts with Copper/Zinc
Superoxide Dismutase (1998) The Journal of Biological Chemistry 273(37):23625-23628
2) Steen V. Petersen et al. The dual nature of human extracellular superoxide dismutase: One
sequence and two structures (2003) PNAS 100(24):13875-13880
3) Tim D. Oury et al. Human extracellular superoxide dismutase is a tetramer composed of
two disulphide-linked dimmers: a simplified, high-yield purification of extracellularsuperoxide dismutase (1996) Biochem .J .317:51-57
4) Tohru Fukai et al. Extracellular superoxide dismutase and cardiovascular disease (2002)
Cardiovascular Research 55:239-249
5) Yoshiaki Furukawa et al. Oxygen-induced maturation of SOD1: a key role for disulfide
formation by the copper chaperone CCS (2004) The EMBO Journal 23:2872-2881
6) Richard W. Strange et al. The Structure of Holo and Metal-dificient Wild-type Human Cu,
Zn Superoxide Dismutase and its Relevance to Familial Amyotrophic Lateral Sclerosis(2003) J. Mol. Biol. 328:877-891
7) Stefan I. Liochev et al. Cross-compartment protection by SOD1 (2005) Free Radical
Biology & Medicine 38:146-147
8) Richard A. Weisiger et al. Superoxide Dismutase (1973) The Journal of Biol. Chem.
248(10):3582-3592

13◈ Ordering Information
For orders, please contact :Young In Frontier Co., Ltd.
Tel : +82-2-2140-3300
Fax: +82-2-2140-3310
E-mail: orders@younginfrontier.com
Address: 11F, Byucksan Digital Valley 5th, Gasan-dong 60-73, Geumcheon-gu, Seoul,
Korea (153-801)
Website: http://www.abfrontier.com
Or, your local distributor.

For technical advice, please contact:
E-mail : orders@younginfrontier.com
Website : http://www.abfrontier.com

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