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  Mouse Endothelin 1（ （ （ （ET-1） ） ） ）
  ELISA Kit  
  
Catalog No. CSB-E05145m
（96T）
 
 
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of mouse
ET-1  concentrations  in  cell  culture  supernates,  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
Endothelin-1（ET-1）, a peptide of 21 amino acid residues, is the most potent
vasoconstrictive  substance  known. Originally  isolated  from  porcine  aortic
endothelial  cells,  ET-1  is  now  known  to  be  one  of  a  family  of  three
mammalian vasoactive peptides that also includes Endothelin-2 （ET-2） and
Endothelin-3 （ET-3）. These related peptides differ from ET-1 at two and six
amino  acid  residue  positions,  respectively.  A  fourth  peptide,  vasoactive
intestinal  contractor  （VIC）,  is    sometimes  classified  as  rat  ET-2.  All
members  of  the  endothelin  family  contain  two  essential  disulfide  bridges
and six conserved amino acid residues at the C-terminus. Additionally, all of
the  endothelin  family  members  are  synthesized  initially  as
prepropolypeptides of approximay 200 amino acid residues encoded by
separate  genes.  These  are  proteolytically  cleaved  to  produce  
biologically-inactive  propolypeptides  of  approximay  40  amino  acid
residues  termed  “big  endothelins”. Big ET-1  is  cleaved  by  the  proteolytic
action  of  a  membrane-bound  metalloprotease  [endothelin-converting
enzyme （ECE-1）] producing the 21 amino acid residue active peptide. The
biochemistry  and  biology  of  the  endothelins  have  been  the  subject  of
several reviews.
PRINCIPLE OF THE ASSAY
The  microtiter  plate  provided  in  this  kit  has  been  pre-coated  with  an
antibody  specific  to  ET-1.  Standards  or  samples  are  then  added  to  the
appropriate  microtiter  plate  wells  with  a  biotin-conjugated  polyclonal
antibody preparation   
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specific for ET-1. and Avidin conjugated  to Horseradish Peroxidase （HRP）
is  added  to  each microplate  well  and  incubated.  Then  a  TMB  （3,3'5,  5'
tetramethyl-benzidine） substrate solution is added to each well. Only those
wells that contain ET-1, 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 ET-1. in the samples is then determined by comparing the
O.D. of the samples to the standard curve.
DETECTION RANGE
1.6  pg/ml-100  pg/ml.  The  standard  curve  concentrations  used  for  the
ELISA’s  were  100  pg/ml,  50  pg/ml,  25  pg/ml,  12.5  pg/ml,  6.2  pg/ml,  3.2
pg/ml,1.6pg/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  mouse  ET-1    No
significant cross-reactivity or interference was observed.
SENSITIVITY
The  minimum  detectable  dose  of  mouse  ET-1  is  typically  less  than  0.8
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 120µl
HRP-avidin  1 x 120µl
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  100  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 （100 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.
OTHER SUPPLIES REQUIRED
  Microplate  reader  capable  of measuring  absorbance  at  450  nm, with
the correction wavelength set at 540 nm or 570 nm. 
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  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 as
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. 
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1.  Add  100µl  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 100µl 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 （200µl） 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  100µl  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 90µl 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.  
8.  Add  50µl  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. 
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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  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
ET-1. 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.
  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. 
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  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  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.
  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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