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Porcine Tumor Necrosis Factor α 
（TNF-α） ELISA Kit
 
 
 
 
 
Catalog No. CSB-E06840p
（96T）
 
 
 
 
 
 
?  This immunoassay kit allows for the in vitro quantitative determination of porcine
TNF-α concentrations in serum, plasma.
?  Expiration date    six months from the date of manufacture
?  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
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INTRODUCTION
The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A,
is a pleiotropic cytokine that plays a central role in inflammation
and apoptosis. It is synthesized as a 26 kDa, type II
transmembrane protein that is 233  amino acids in length. It
contains a 30 amino acid （aa） cytoplasmic domain, a 26 aa
transmembrane segment, and a 177 aa extracellular region.
TNF-αis assembled intracellularly to form a transmembrane,
non-covalently-linked homotrimeric protein. The 157 aa residue
soluble form of TNF-α （sTNF-αis released from the C-terminus of
the  transmembrane protein through the activity of
TNF-α-converting enzyme （TACE）, a membrane -bound
disintegrin metalloproteinase. Human cells known to express
TNF-αinclude B cells, colonic columnar epithelial cells, NK and
CD3+CD56+ hepatic natural T  cells, macrophages, monocytes
and monocyte-derived dendritic cells, CD4+ and CD8+ T cells,
mast cells, neutrophils, keratinocytes, plasma cells, and
adipocytes. 
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
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an antibody specific to TNF-α. Standards or samples are then
added to the appropriate microtiter plate wells with a Horseradish
Peroxidase （HRP）-conjugated monoclonal antibody preparation
specific for TNF-α and incubated. Then substrate solution A and
B are added to each well. Only  those wells that contain TNF-α,
HRP-conjugated antibody will exhibit a change in color. The
enzyme-substrate reaction is terminated by the addition of a
sulphuric acid solution and the color change is measured
spectrophotometrically at a wavelength of 450 nm ± 2 nm. The
concentration of TNF-α in the samples is then determined by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
The standard curve concentrations used for the ELISA’s were 25
ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.125 ng/ml, 1.25 ng/ml,
0.625ng/ml.
SPECIFICITY
This assay recognizes recombinant and natural porcine TNF-α.
No significant cross-reactivity or interference was observed.
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SENSITIVITY
The minimum detectable dose of porcine TNF-α is typically less
than 0.312 ng/ml. The sensitivity of this assay, or Lower Limit of
Detection （LLD） was defined as the lowest concentration that
could be differentiated from zero.
MATERIALS PROVIDED
Reagent    Quantity
Assay plate  1
Standard（S1-S6）  6
HRP-conjugate  1 x 6 ml
Wash Buffer      
1 x 15 ml
 （20×concentrate）
Substrate A  1 x 6 ml
Substrate B  1 x 6 ml
Stop Solution      1x 6 ml
 
Standard  S1  S2  S3  S4  S5  S6
Concentration （ng/ml）  0.625 1.25 3.125 6.25 12.5  25
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STORAGE
1. Unopened test kits should be stored at 2-8?C upon receipt
and the microtiter plate should be kept in a sealed bag. The
test kit may be used throughout the expiration date of the kit,
provided it is stored as prescribed above. Refer to the
package label for the expiration date.
2. Opened test plate should be stored at 2-8?C in the aluminum
foil bag with desiccants to minimize exposure to damp air. The
kits will remain stable until the expiring date shown, provided it
is stored as prescribed above.  
3. A microtiter plate reader with a bandwidth of 10 nm or less
and an optical density range of 0-3 OD or greater at 450nm
wavelength is acceptable for use in absorbance
measurement.
REAGENT PREPARATION
1. Bring all reagents and plate to room temperature  for at least
30 minutes before use. Unused wells need store at 2-8°C and
avoid sunlight.
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2. Wash Buffer   If crystals have formed in the concentrate,
warm to room temperature and mix gently until the crystals
have compley dissolved. Dilute 15 ml of Wash Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material.
OTHER SUPPLIES REQUIRED
?  Microplate reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
?  Pipettes and pipette tips.
?  Deionized or distilled water.
?  Squirt bottle, manifold dispenser, or automated microplate
washer.
?  An incubator which can provide stable incubation conditions
up to 37°C±0.5°C.
SAMPLE COLLECTION AND STORAGE
?  Serum  Use a serum separator tube （SST） and allow
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samples to clot for 30 minutes before centrifugation for 15
minutes at 1000 g. Remove serum and assay immediay or
aliquot and store samples at  -20°C. Centrifuge the sample
again after thawing before  the assay. Avoid repeated
freeze-thaw cycles.
?  Plasma   Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes of collection. Assay immediay or aliquot and
store samples at -20°C. Centrifuge the sample again after
thawing before the assay. Avoid repeated freeze-thaw cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate.
All the reagents should be added directly to the  liquid level in the well. The
pipette should avoid contacting the inner wall of the well.
1.  Set a Blank well without any solution. Add 100μl of Standard
or Sample per well. Standard need test in duplicate.  
2. Add 50μl of HRP-conjugate to each well （not to Blank well）.
Mix well and then incubate for 2 hours at 37°C.  
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3.  Complete remove the liquid. Then fill each well with Wash
Buffer （about 200μl）, stay for 10 seconds and Spinning.
Repeat the process for a total  of three washes. Complete
removal of liquid at each step is essential to good
performance. After the last wash, remove any remaining
Wash Buffer by aspirating or decanting. Invert the plate and
blot it against clean paper towels.
4. Add 50μl of Substrate A and 50μl of Substrate B to each
well, mix well. Incubate for 15 minutes at 37°C. Keeping the
plate away from drafts and other temperature fluctuations in
the dark.
5. Add 50μl of Stop Solution to each well. If color change does
not appear uniform, gently tap the plate to ensure thorough
mixing.
6.  Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
Using the professional soft "Curve Exert  1.3" to make a standard curve is
recommended, which can be downloaded from our web.
Average the duplicate readings for each standard, control, and
  8
sample and subtract the average zero standard optical density.
Create a standard curve by reducing the data using computer
software capable of generating a four parameter logistic （4-PL）
curve-fit. As an alternative, construct a standard curve by plotting
the mean absorbance for each standard on the y-axis against
the concentration on the x-axis and draw a best fit curve through
the points on the graph. The data may be linearized by plotting
the log of the TNF-α concentrations versus the log of the O.D.
and the best fit line can be determined by regression analysis.
This procedure will produce an adequate but less precise fit of
the data. If samples have been diluted, the concentration read
from the standard curve must be multiplied by the dilution factor.
LIMITATIONS OF THE PROCEDURE
?  The kit should not be used beyond the expiration date on the
kit label.
?  Do not mix or substitute reagents with those from other lots or
sources.
?  If samples generate values higher than the highest standard,
dilute the samples with the appropriate Diluent and repeat the
assay.
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? Any variation in operator, pipetting technique, washing
technique, incubation time or  temperature, and kit age can
cause variation in binding.
?  This assay is designed to eliminate interference by soluble
receptors, binding proteins,  and other factors present in
biological samples. Until all factors have been tested in the
Immunoassay, the possibility of interference cannot be
excluded.
TECHNICAL HINTS
?  Centrifuge vials before opening to collect contents.
?  When mixing or reconstituting protein solutions, always avoid
foaming.
?  To avoid cross-contamination, change pipette  tips between
additions of each standard level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
?  When using an automated plate washer, adding a 30 second
soak period following the addition of wash buffer, and/or
rotating the plate 180 degrees between wash steps may
improve assay precision.
  10
  11
?  To ensure accurate results, proper adhesion of plate sealers
during incubation steps is necessary.
?  Substrate Solution should remain colorless or light blue until
added to the plate. Keep Substrate Solution protected from
light. Substrate Solution should change from colorless or light
blue to gradations of blue.
?  Stop Solution should be added to the plate in the same order
as the Substrate Solution. The color developed in the wells
will turn from blue to yellow upon addition of the Stop Solution.
Wells that are green in color indicate that the Stop Solution
has not mixed thoroughly with the Substrate Solution.