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Mouse Tumor Necrosis Factor α
（TNF-α）ELISA KIT
 
 
 
Catalog No. CSB-E04741m
（96 tests）
 
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of mouse
TNF-α concentrations in serum, plasma and other biological fluids.
  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  an
antibody  specific  to TNF-α. Standards  or  samples  are  then  added  to  the
appropriate  microtiter  plate  wells  with  a  biotin-conjugated  polyclonal
antibody  preparation  specific  for  TNF-α  and  Avidin  conjugated  to
Horseradish  Peroxidase  （HRP）  is  added  to  each  microplate  well  and 
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incubated. Then a TMB （3,3'5, 5'  tetramethyl-benzidine） substrate solution
is  added  to  each  well.  Only  those  wells  that  contain  TNF-α,
biotin-conjugated  antibody  and  enzyme-conjugated  Avidin  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
15.6  pg/ml  -1000  pg/ml.  The  standard  curve  concentrations  used  for  the
ELISA’s were 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5pg/ml, 31.2
pg/ml, 15.6 pg/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  mouse  TNF-α.  No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum  detectable  dose  of mouse TNF-α  is  typically  less  than  3.9
pg/ml.
The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined
as the lowest protein concentration that could be differentiated from zero. 
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MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standard  2
Sample Diluent      1 x 20 ml
Biotin-antibody Diluent      1 x 10 ml
HRP-avidin Diluent        1 x 10 ml
Biotin-antibody      1 x 120l
HRP-avidin  1 x 120l
Wash Buffer      
1 x 20 ml
  （25×concentrate）
TMB Substrate      1 x 10 ml
Stop Solution      1 x 10 ml
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. 
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REAGENT PREPARATION
Bring all reagents to room temperature before use.  
1.  Wash Buffer    If crystals have  formed  in  the concentrate, warm up  to
room  temperature  and  mix  gently  until  the  crystals  have  compley
dissolved. Dilute 20 ml of Wash Buffer Concentrate  into deionized or
distilled water to prepare 500 ml of Wash Buffer.
2.  Standard    Centrifuge  the  standard  vial  at  6000-10000rpm  for  30s.
Reconstitute  the  Standard  with  1.0  ml  of  Sample  Diluent.  This
reconstitution  produces  a  stock  solution  of  1000  pg/ml.  Allow  the
standard to sit for a minimum of 15 minutes with gentle agitation prior to
making  serial  dilutions.  The  undiluted  standard  serves  as  the  high
standard  （1000  pg/ml）.  The  Sample  Diluent  serves  as  the  zero
standard  （0 pg/ml）. Prepare  fresh  for each assay. Use within 4 hours
and discard after use.
3.  Biotin-antibody    Centrifuge  the  vial  before  opening.  Dilute  to  the
working  concentration  using  Biotin-antibody  Diluent（1:100）,
respectively.
4.  HRP-avidin    Centrifuge  the vial before opening. Dilute  to  the working
concentration using HRP-avidin Diluent（1:100）, respectively.
Precaution: The Stop Solution provided with  this kit  is an acid solution. Wear
eye, hand, face, and clothing protection when using this material. 
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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 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. 
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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.  Add  100l  of  Standard,  Blank,  or  Sample  per  well.  Cover  with  the
adhesive strip. Incubate for 2 hours at 37°C.  
2.  Remove the liquid of each well, don’t wash.  
3.  Add 100l of Biotin-antibody working solution  to each well.  Incubate
for  1  hour  at  37°C.  Biotin-antibody  working  solution  may  appear
cloudy. Warm  up  to  room  temperature  and  mix  gently  until  solution
appears uniform.
4.  Aspirate each well and wash,  repeating  the process  three  times  for a
total of three washes. Wash: Fill each well with Wash Buffer （200l） and
let  it  stand  for  2 minutes,  then  remove  the  liquid  by  flicking  the  plate
over a sink. The remaining drops are removed by patting the plate on a
paper  towel.  Complete  removal  of  liquid  at  each  step  is  essential  to
good performance.
5.  Add  100l  of  HRP-avidin  working  solution  to  each  well.  Cover  the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
6.  Repeat the aspiration and wash three times as step 4.
7.  Add 90l of TMB Substrate to each well. Incubate for 10-30 minutes at
37°C.  Keeping  the  plate  away  from  drafts  and  other  temperature
fluctuations in the dark.   
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8.  Add  50l  of  Stop  Solution  to  each  well  when  the  first  four  wells
containing the highest concentration of standards develop obvious blue
color.  If color change does not appear uniform, gently  tap  the plate  to
ensure thorough mixing.
9.  Determine  the optical density of each well within 30 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 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  x-axis
against the concentration on the y-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. 
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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.
  It is important that the Standard Diluent selected for the standard curve
be consistent with the samples being assayed.
  If samples generate values higher than the highest standard, dilute the
samples with the appropriate Standard Diluent and repeat the assay.
  Any  variation  in  Standard  Diluent,  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. 
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  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.
  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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