Human Neuron Specific Enolase (NSE) ELISA

Catalog # LF-EK0169 (1 kit bundle)
Catalog # LF-EK0170 (4 kits bundle)
Catalog # LF-EK0171 (10 kits bundle)
Catalog # LF-EK0172 (20 kits bundle)

Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection of
human Neuron Specific Enolase(NSE)

For research use only
Not for diagnostic or therapeutic procedures

Contents
1. Introduction ···················································································· 3
2. Principles of Method ······································································ 3
3. Intended Use ··················································································· 4
4. Storage and Stability ····································································· 4
5. Chemical Hazard ··········································································· 4
6. Kit Contents ···················································································· 5
7. Materials Required But Not Provided ········································· 6
8. Reagent preparation ······································································ 6
1) Human Neuron Specific Enolase standard ································· 6
2) Secondary Antibody ································································· 6
3) AV-HRP ·················································································· 7
4) Washing buffer ········································································ 7
9. Assay Procedure ············································································· 8
10. Characteristics ··············································································· 9
1) Typical result ··········································································· 9
2) Sensitivity ··············································································· 10
3) Specificity ··············································································· 11
4) Precision ················································································· 11
5) Recovery ················································································· 11
11. Troubleshooting ·············································································· 12
12. Reference ························································································ 13
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1. Introduction
Enolase (2-phosphogly-cerate hydrolyase or phosphopyruvate hydrates) is a glycolytic
enzyme that catalyzes the dehydration and conversion of 2-phosphoglycerate to
phosphoenolpyruvate. It comprises three distint subunits, α, β and γ. The γγ and αγ dimeric
forms of enolase, referred to as neuron-specific enolase(NSE), are localized mainly in neurons
and neuroectodermal tissue. NSE has a high stability in biological fluids and can easily diffuse
to the extracellular medium and cerebrospinal fluid(CSF) when neuronal membranes are injured.
NSE is used clinically as a sensitive and useful marker of neuronal damage in several
neurological disorders including stroke, hypoxic brain damage, status epilepticus, CreutzfeldtJakob disease, and herpetic encephalitis.
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 NSE. Samples are pippetted into these wells. Nonbound NSE and
other components of the sample should be removed by washing, then biotin-conjugated
monoclonal antibody specific to NSE added. In order to quantitatively determine the amount of
NSE 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
NSE.
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3. Intended Use
The AbFrontier human neuron specific enolase(human NSE) ELISA kit is to be used for the
in vitro quantitative determination of human NSE in human serum, human plasma, cell
lysate and buffered solution. The assay will recognize native NSE.
This kit has been configured for research use only and is not to be used in
diagnostic procedures.
4. Storage and Stability
All kit components of this kit are stable at 2 to 8℃. Any unused reconstituted standard
should be discarded or frozen at -70℃. 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 of
water.
- All reagents containing Sodium Azide also contain Thimerosal as a preservative.
Thimerosal contains Hg thus should be handled with great care.
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6. Kit Contents
Contents Number Volume
96 Well Plate 1 (in aluminum foil bag with desiccant)
Incubation Buffer 1 30ml
Washing Buffer 1 (10X) 100ml
Standard Protein 1 Glass vial (lyophilized)
Standard/Sample Dilution Buffer 1 25ml
Secondary Antibody 1 Glass vial (lyophilized)
AV-HRP 1 150ul
Secondary Antibody/AV-HRP
Dilution Buffer
1 25ml
Substrate (TMB) 1 20ml
Stop Solution 1 20ml
Protocol booklet 1
Plate sealers 2
① 96 Well Plate
: Human NSE microtiter plate, one plate of 96 wells (16well strip x 6).
A plate using break-apart strips coated with a mouse monoclonal antibody specific to
human neuron specific enolase.
② Standard Protein
: Native human neuron specific enolase.
③ Secondary Antibody
: Biotin labeled mouse anti human neuron specific enolase antibody.
④ AV-HRP
: Avidin linked Horseradish Peroxidase (HRP, enzyme)
⑤ Substrate (Stabilized chromogen)
: Tetramethylbenzidine (TMB) solution
⑥ Stop Solution
: 1N solution of sulphuric acid (H2SO4).
⑦ Plate sealer
: Adhesive sheet.
 Do not mix or interchange different reagents from various kit lots.
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7. Materials Required But Not Provided
① Microtiter plate reader capable of measurement at or near 450nm.
② Calibrated, adjustable precision pipettes, preferably with disposable plastic tips (A
manifold multi-channel pipette is desirable for large assays.)
③ Distilled or deionized water
④ Data analysis and graphing software
⑤ Vortex mixer
⑥ Polypropylene tubes for diluting and aliquoting standard
⑦ Absorbent paper towels
⑧ Calibrated beakers and graduated cylinders of various sizes
8. Reagent Preparation
1) Human Neuron specific enolase standard
Reconstitute the human NSE standard to 0.5ug/ml by adding 1ml of Standard/Sample
Dilution Buffer into the standard protein glass vial containing lyophilized human NSE
protein. Swirl or mix gently, and allow to sit for 5 minutes to ensure complete
reconstitution.
Standard Add Into
80ng/ml 160ul of the std.(0.5ug/ml) 840ul of the Standard/Sample Dilution Buffer
40ng/ml 80ul of the std.(0.5ug/ml) 920ul of the Standard/Sample Dilution Buffer
20ng/ml 40ul of the std.(0.5ug/ml) 960ul of the Standard/Sample Dilution Buffer
10ng/ml 20ul of the std.(0.5ug/ml) 980ul of the Standard/Sample Dilution Buffer
5ng/ml 10ul of the std.(0.5ug/ml) 990ul of the Standard/Sample Dilution Buffer
2.5ng/ml 5ul of the std.(0.5ug/ml) 995ul of the Standard/Sample Dilution Buffer
1.25ng/ml 2.5ul of the std.(0.5ug/ml) 997.5ul of the Standard/Sample Dilution Buffer
0ng/ml 1.0ml of the Standard/Sample Dilution Buffer
2) Secondary Antibody
100X secondary antibody solution can be made by adding 150 ㎕ secondary
antibody/AV-HRP dilution buffer in the vial.
1. Equilibrate to room temperature, mix gently.
2. Mix 20ul Secondary Antibody concentrated solution (100X) + 2ml Secondary
Antibody/AV-HRP dilution buffer. (Sufficient for one 16-well strip, prepare more if
necessary)
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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 20ul AV-HRP concentrated solution (100X) + 2ml Secondary Antibody/AV-HRP
dilution buffer. (Sufficient for one 16-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 1 volume Wash buffer concentrate solution (10X) + 9 volumes of deionized water.
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 300ul of “Working Washing Buffer”.
Let soak for 1 to 3 minutes and then all residual wash-liquid must be drained from the
wells 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 venipuncture. 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.
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9. Assay Procedure
 Allow all reagents to reach room temperature before use. Gently mix all liquid reagents
prior 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 300ul 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 100ul of the standard to the appropriate microtiter wells. Add
100ul 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 100ul of samples to each wells.
6) Cover the plate with the plate cover and incubate for 2 hours at 37℃.
7) Thoroughly aspirate or decant the solution from the wells. Wash the wells 3 times (See
“Directions for washing”).
8) Pipette 100ul 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 100ul “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 100ul 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
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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 100ul of Stop Solution to each well. The solution in the wells should change from
blue to yellow.
17) Read the absorbance of each well at 450nm. 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 these
points to construct the standard curve.
19) Read the human NSE concentrations for the unknown samples and controls from the
standard 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
human NSE (ng/ml)
Optical Density
(at 450nm)
0 0.093
1.25 0.097
2.5 0.109
5 0.151
10 0.273
20 0.634
40 1.463
80 2.728
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< Limitations >
 Do not extrapolate the standard curve beyond the 80ng/ml standard point.
 Other buffers and matrices have not been investigated.
 The rate of degradation of native human neuron specific enolase in various matrices has
not been investigated.
(TMB reaction incubate at room temperature for 5min)
2) Sensitivity
The minimal detectable dose of human NSE was calculated to be 0.15ng/ml, by
subtracting two standard deviations from the mean of 12 zero standard replicates (ELISA
buffer, S0) and intersecting this value with the standard curve obtained in the same
calculation.
N 1 2 3 4 5 6 7 8 9 10 11 12
ZERO 0.09 0.088 0.089 0.091 0.09 0.096 0.093 0.102 0.096 0.094 0.094 0.097
3) Specificity
The following substances were tested and found to have no cross-reactivity: human
serum albumin, human non neuronal enolase, human alpha fetoprotein, human prostate
specific antigen(PSA), human hemoglobin, human VDBP(vitamin D binding protein)
Average SD LLD
LLD
mean(ng/ml)
0.093 0.00403 0.101394 2.447
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4) Precision
① Within-Run (Intra-Assay)
(n=6)
Mean (ng/ml) SD (ng/ml) CV (%)
7.44 0.26 3.47
17.46 0.84 4.81
43.03 2.22 5.17
79.58 3.41 4.28
② Between-Run (Inter-Assay)
(n=4)
Mean (ng/ml) SD (ng/ml) CV (%)
7.15 0.43 6.07
17.31 0.74 4.25
42.87 2.20 5.13
79.43 3.47 4.37
5) Recovery
Recovery on addition is 75.36~108.38% (mean 91.94%)
Analyte addded
(ng/ml)
Serum A
(ng/ml)
Recovery
(%)
10.0 7.64 86.03
20.0 14.44 75.36
40.0 34.44 89.90
80.0 86.80 108.38


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11. Troubleshooting
Problem Possible Cause Solution
High signal and background
in all wells
• Insufficient washing • Increase number of washes
• Increase time of soaking
between in wash
• 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
No signal
• Reagent added in incorrect
order, or incorrectly prepared
• Review protocol
• Standard has gone bad
(If there is a signal in the
sample wells)
• Check the condition of stored
standard
• Assay was conducted from
an incorrect starting point
• Reagents allows to come to
20~30℃ before performing
assay
Too much signal – whole
plate turned uniformly blue
• Insufficient washing
– unbound AV-HRP remaining
• Increase number of washes
carefully
• Too much AV-HRP • Check dilution
• Plate sealer or reservoir
reused, resulting in presence
of residual AV-HRP
• Use fresh plate sealer and
reagent reservoir for each
step
Standard curve achieved but
poor discrimination between
point
• Plate not developed long
enough
• Increase substrate solution
incubation time
• Improper calculation of
standard curve dilution
• Check dilution, make new
standard curve
No signal when a signal is
expected, but standard curve
looks fine
• Sample matrix is masking
detection
• More diluted sample
recommended
Samples are reading too high,
but standard curve is fine
• Samples contain protein
levels above assay range
• Dilute samples and run
again
Edge effect
• Uneven temperature around
work surface
• Avoid incubating plate in
areas where environmental
conditions vary
• Use plate sealer
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12.Reference
1. Fletcher L. et al. (1976) Biochim. Biophys. Acta. 452(1), 245-252
2. Lima J.E. et al. (2004) J. Neurol. Sci. 217(1), 31-35
3. Suzuki Y. et al. (1999) Neurology 53(8), 1761-1764
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Tel : +82-1577-2684
Fax: +82-31-460-9410
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:
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