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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, 6.2 ng/ml, 3.2 ng/ml, 1.6 ng/ml, 0.8ng/ml.
SPECIFICITY
This assay  recognizes  recombinant and natural porcine TNF-α.
No significant cross-reactivity or interference was observed. 
  4
SENSITIVITY
The minimum detectable dose of porcine TNF-α is typically less
than  0.4  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）  0.8  1.6  3.2  6.2  12.5  25 
  5
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 100l of Standard
or Sample per well. Standard need test in duplicate.  
2.  Add 50l 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  200l）,  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 50l of Substrate A and 50l of Substrate B  to each
well, mix well.  Incubate for 15 minutes at 37°C. Ke eping the
plate away  from drafts and other  temperature  fluctuations  in
the dark.
5.  Add 50l 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 
  9
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. 
  10
  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
Quantikine  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. 
  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. 
 
 
 

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