Human NGF/NGFβ ELISA

Catalog # LF-EK50285 (1 kit)
Catalog # LF-EK50286 (4 kits bundle) 

Sandwich Enzyme-Linked Immunosorbent Assay for Quantitative Detection of
human NGF/NGFβ
For research use only
Not for diagnostic or therapeutic purposes

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
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1) Sample Preparation and Storage
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2) Sample Dilution Guideline ···················································· 6
3) Reagent Preparation and Storage
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9. Assay Procedure
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10. Characteristics
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1) Typical result
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2) Sensitivity
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3) Specificity
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11. Troubleshooting
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12. Reference
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1. Introduction

Nerve growth factor (NGF) is a polypeptide involved in the regulation of growth and
differentiation of sympathetic and certain sensory neurons. NGF is thought to have a profound
effect on the development and maintenance of sympathetic and embryonic sensory neurones.
NGF activity isolated from the male mouse submaxillary gland (MSG) consists of three types of
subunits, alpha, beta and gamma, which specifically interact to form a 7S, approximately
130,000-molecular weight (Mr) complex. The 7S complex contains two identical 118-amino
acid beta-chains, which are solely responsible for the nerve growth-stimulating activity of
NGF.1
NGF, which is expressed by inflammatory cells and effects changes that lead to increased
neural responsiveness, could be a pivotal mediator in allergic rhinitis.2
The standard product
used in this kit is human 2.5S NGF, which is a dimmer linking with two polypeptide chains of
120 amino acids.
2. Principles of Method
AbFrontier’s human NGF ELISA Kit was based on standard sandwich enzyme-linked
immune-sorbent assay technology. Human NGF specific-specific monoclonal antibodies were
precoated onto 96-well plates. The human specific detection polyclonal antibodies were
biotinylated. The test samples and biotinylated detection antibodies were added to the wells
subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase
Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP
substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to
produce a blue color product that changed into yellow after adding acidic stop solution. The
density of yellow is proportional to the human NGF amount of sample captured in plate..
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3. Intended Use
The AbFrontier human NGF ELISA kit is to be used for the in vitro quantitative determination
of human NGF in sera, plasma, body fluids, tissue lysates or cell culture supernates.
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 -20℃. 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)
Standard Protein 2 10ng/tube
Secondary Antibody 1 130 ul
Avidin-Biotin-Peroxidase Complex
(ABC)
1 130 ul
Sample diluent Buffer 1 30 ml
Antibody diluent buffer 1 12 ml
ABC diluent buffer 1 12 ml
TMB color developing agent 1 10 ml
TMB stop solution 1 10 ml
① 96 Well Plate
: Human NGF microtiter plate, one plate of 96 wells.
A plate using break-apart strips coated with a monoclonal antibody specific to human
NGF.
② Standard Protein
: Lyophilized human NGF.
③ Secondary Antibody
: Biotin labeled anti human NGF antibody.
④ AV-HRP
: Avidin-Biotin-Peroxidase Complex (ABC)
⑤ Substrate (Stabilized chromogen)
: Tetramethylbenzidine (TMB) solution
⑥ Stop Solution
: 1 N solution of sulfuric acid (H2SO4)
Notice for Application of Kit
1． Before using Kit, spin tubes and bring down all components to bottom of tube.
2． Duplicate well assay was recommended for both standard and sample testing.
3． Don’t let 96-well plate dry, dry plate will inactivate active components on plate.
4． In order to avoid marginal effect of plate incubation due to temperature difference
(reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB
solution will be pre-warmed in 37℃ for 30 min before using.
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7. Materials Required But Not Provided
① Microtiter plate reader in standard size.
② Automated plate washer.
③ Distilled or deionized water
④ Calibrated, adjustable precision pipettes, preferably with disposable plastic tips (A
manifold multi-channel pipette is desirable for large assays.)
⑤ Data analysis and graphing software
⑥ Vortex mixer
⑦ Polypropylene tubes for diluting and aliquoting standard
⑧ Absorbent paper towels
⑨ Calibrated beakers and graduated cylinders of various sizes
⑩ Washing buffer (neutral PBS or TBS). Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g
Nacl; 450µl of purified acetic acid or 700µl of concentrated hydrochloric acid to 1000ml
H2
O and adjust pH to 7.2-7.6. Finally, adjust the total volume to 1L.
Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na2
HPO4 and 0.2g
NaH2
PO4 to 1000ml distilled water and adjust pH to 7.2-7.6. Finally, adjust the total
volume to 1L.
8. Reagent Preparation
1) Sample Preparation and Storage
Store samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot and
freeze samples at -20°C.
Avoid repeated freeze-thaw cycles.
o Cell culture supernate, tissue lysate or body fluids: Remove particulates by
centrifugation, analyze immediately or aliquot and store at -20°C
o Serum: Allow the serum to clot in a serum separator tube (about 4 hours) at room
temperature. Centrifuge at approximately 2000 X g for 20 min. Analyze the
serum immediately or aliquot and store frozen at -20°C.
o Plasma: Collect plasma using heparin as an anticoagulant. Centrifuge for 10 min at
1000 x g within 30 min of collection. Analyze immediately or aliquot and store
frozen at -20°C.
2) Sample Dilution Guideline
The user needs to estimate the concentration of the target protein in the sample and select
a proper dilution factor so that the diluted target protein concentration falls near the
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middle of the linear regime in the standard curve. Dilute the sample using the provided
diluent buffer. The following is a guideline for sample dilution. Several trials may be
necessary in practice. The sample must be well mixed with the diluents buffer.
o High target protein concentration (10-100ng/ml). The working dilution is 1:100.
i.e. Add 1 µl sample into 99 µl sample diluent buffer.
o Medium target protein concentration (1-10ng/ml). The working dilution is 1:10.
i.e. Add 10 µl sample into 90 µl sample diluent buffer.
o Low target protein concentration (15.6-1000pg/ml). The working dilution is 1:2.
i.e. Add 50 µl sample to 50 µl sample diluent buffer.
o Very Low target protein concentration (≤15.6pg/ml). No dilution necessary, or
the working dilution is 1:2.
3) Reagent Preparation and Storage
A. Reconstitution of the human NGF standard： NGF standard solution should be
prepared no more than 2 hours prior to the experiment. Two tubes of NGF standard (10ng
per tube) are included in each kit. Use one tube for each experiment.
a. 10,000pg/ml of human NGF standard solution: Add 1 ml sample diluent buffer into
one tube, keep the tube at room temperature for 10 min and mix thoroughly.
b. 1000pg/ml of human NGF standard solution: add 0.1m of the above 10ng/ml NGF
standard solution into 0.9ml sample diluent buffer and mix thoroughly.
c. 500pg/ml→15.6pg/ml of human NGF standard solutions: Label 6 Eppendorf tubes
with 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml,
respectively. Aliquot 0.3 ml of the sample diluent buffer into each tube. Add
0.3 ml of the above 1000pg/ml NGF standard solution into 1st tube and mix.
Transfer 0.3 ml from 1st tube to 2nd tube and mix. Transfer 0.3 ml from 2nd
tube to 3rd tube and mix, and so on.
Note: The standard solutions are best used within 2 hours. The 10 ng/ml standard
solution may be stored at 4°C for up to 12 hours, or at -20°C for up to 48 hours.
Avoid repeated freeze-thaw cycles.
B. Preparation of biotinylated anti-human NGF antibody working solution: The solution
should be prepared no more than 2 hours prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2
ml more than total volume)
b. Biotinylated anti-human NGF antibody should be diluted in 1:100 with the
antibody diluent buffer and mixed thoroughly.
C. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The
solution should be prepared no more than 1 hour prior to the experiment.
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a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2
ml more than total volume)
b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the
ABC dilution buffer and mixed thoroughly.
9. Assay Procedure
The ABC working solution and TMB color developing agent must be kept warm at 37°C
for 30 min before use. When diluting samples and reagents, they must be mixed
completely and evenly. Standard NGF detection curve should be prepared for each
experiment. The user will decide sample dilution fold by crude estimation of NGF amount
in samples.
1. Aliquot 0.1ml per well of the 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml,
31.3pg/ml, 15.6pg/ml human NGF standard solutions into the precoated 96-well plate.
Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add 0.1ml of
each properly diluted sample of human serum, plasma, body fluids, tissue lysates or cell
culture supernatants to each empty well. See “Sample Dilution Guideline” above for
details. We recommend that each human NGF standard solution and each sample is
measured in duplicate.
2. Seal the plate with the cover and incubate at 37°C for 90 min.
3. Remove the cover, discard plate content, and blot the plate onto paper towels or other
absorbent material. Do NOT let the wells completely dry at any time.
4. Add 0.1ml of biotinylated anti-human NGF antibody working solution into each well
and incubate the plate at 37°C for 60 min.
5. Wash plate 3 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer
stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper
towels or other absorbent material. (Plate Washing Method: Discard the solution in the
plate without touching the side walls. Blot the plate onto paper towels or other absorbent
material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat
this process two additional times for a total of THREE washes. Note: For automated
washing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfilling
wells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent
material.)
6. Add 0.1ml of prepared ABC working solution into each well and incubate the plate at
37°C for 30 min.
7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer
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stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper
towels or other absorbent material. (See Step 5 for plate washing method).
8. Add 90 µl of prepared TMB color developing agent into each well and incubate plate at
37°C for 20-25min (Note: For reference only, the optimal incubation time should be
determined by end user. And the shades of blue can be seen in the wells with the four
most concentrated human NGF standard solutions; the other wells show no obvious
color).
9. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow
immediately.
10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding
the stop solution.
For calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero
well). The standard curve can be plotted as the relative O.D.450 of each standard solution
(Y) vs. the respective concentration of the standard solution (X). The human NGF
concentration of the samples can be interpolated from the standard curve.
Note: if the samples measured were diluted, multiply the dilution factor to the
concentrations from interpolation to obtain the concentration before dilution.
Summary
1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash.
2. Add biotinylated antibodies and incubate the plate at 37°C for 60 min. Wash plate
3 times with 0.01M TBS.
3. Add ABC working solution and incubate the plate at 37°C for 30 min. Wash plate 5
times with 0.01M TBS.
4. Add TMB color developing agent and incubate the plate at 37°C in for 20-25 min.
5. Add TMB stop solution and read.
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10. Characteristics
1) Typical result
Typical Data Obtained from Human NGF
(TMB reaction incubate at 37℃ for 20 min)
Standard
Human NGF (pg/ml)
Optical Density
(at 450nm)
0 0.049
15.6 0.101
31.3 0.174
62.5 0.272
125 0.436
250 0.975
500 1.381
1000 2.210
Typical Human NGF ELISA kit Standard Curve
This standard curve was generated at AbFrontier for demonstration purpose only. A
standard curve must be run with each assay.
2) Sensitivity: < 1pg/ml
3) Specificity: No detectable cross-reactivity with any other cytokine.
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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) Ullrich, A.; Gray, A.; Berman, C.; Dull, T. J. Human beta-nerve growth factor gene sequence
highly homologous to that of mouse. Nature 303: 821-825, 1983.
2) Sanico, A. M.; Stanisz, A. M.; Gleeson, T. D.; Bora, S.; Proud, D.; Bienenstock, J.; Koliatsos,
V. E.; Togias, A. Nerve growth factor expression and release in allergic inflammatory disease of
the upper airways. Am. J. Resp. Crit. Care Med. 161: 1631-1635, 2000.

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Ordering Information

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Young In Frontier Co., Ltd.
Tel : +82-1577-2684
Fax: +82-2-2140-3330
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