Porcine Cortisol ELISA Kit
 
 
Catalog No. CSB-E06811p
（96T）
 
 
 
 
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  quantitative  determination  of  porcine
Cortisol 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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PRINCIPLE OF THE ASSAY
The microtiter  plate  provided  in  this  kit  has  been  pre-coated with  a
goat-anti-rabbit  antibody. Standards or  samples  are  then  added  to  the
appropriate microtiter plate wells with a HRP-conjugated Cortisol and
antibody  preparation  specific  for  Cortisol  and  incubated.  Then
substrate  solutions  are  added  to  each  well.  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 Cortisol in the samples is then
determined  by  comparing  the  O.D.  of  the  samples  to  the  standard
curve.
DETECTION RANGE
0.31 ng/ml-80 ng/ml. The  standard  curve  concentrations used  for  the
ELISA’s were 80 ng/ml, 20 ng/ml, 5 ng/ml, 1.25 ng/ml, 0.31 ng/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  porcine  Cortisol. No 
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significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of porcine Cortisol is typically less than
0.2 ng/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.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standards （S1-S5）  5 x 0.5 ml  
HRP-conjugate  1 x 6 ml
Antibody  1 x 6 ml
Wash Buffer      
1 x 15 ml
  （20×concentrate）
Substrate A  1 x 7 ml
Substrate B  1 x 7 ml
Stop Solution      1 x 7 ml
 
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Standard    S1  S 2  S3  S4  S5
Concentration
（ng/ml）
0.31    1.25    5  20  80  
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 and  avoid  ℃
  5
sunlight.
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  samples  to
clot for 30 minutes before centrifugation for 15 minutes at 1000 g. 
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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  50µl  of  Standard  or
Sample per well. Standard need test in duplicate.  
2.  Add 50µl of HRP-conjugate to each well （not to Blank well）, then
50µl Antibody to each well. Mix well and then incubate for 1 hour
at 37°C.   
  7
3.  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 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, Blank,  and  sample
and  subtract  the optical density of Blank. Create a  standard curve by
reducing the data using computer software capable of generating a four 
  8
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 Cortisol 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.
  Any variation in operator, pipetting technique, washing technique,
incubation  time or  temperature, and kit age can cause variation  in
binding. 
  9
  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.
  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. Sealers can not be reused.
  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 
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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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