Porcine Vascuolar cell adhesion
molecule 1,VCAM-1 ELISA Kit
 
 
 
 
 
Catalog No. CSB-E06825p
（96 tests）
 
 
 
 
 
 
  This  immunoassay kit allows  for  the  in vitro  rapid detection of porcine VCAM-1
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.
 
 
  2
PRINCIPLE OF THE ASSAY
This  assay  employs  the  competitive  inhibition  enzyme
immunoassay technique. A antibody specific to VCAM-1/CD106
has  been  pre-coated  onto  a microplate. Standards  or  samples
are  added  to  the  appropriate  microtiter  plate  wells  with
biotin-conjugated VCAM-1/CD106 and  incubated. A competitive
inhibition  reaction  is  launched  between  VCAM-1/CD106
（Standards  or  samples）  and Biotin-conjugated VCAM-1/CD106
with  the  pre-coated  antibody  specific  for  VCAM-1/CD106.  The
more amount of VCAM-1/CD106  in samples,  the  less antibody
bound  by  Biotin-conjugated  VCAM-1/CD106.  Then  Avidin
conjugated  to Horseradish Peroxidase  （HRP）  is added  to each
microplate  well  and  incubated.  The  substrate  solutions  are
added  to  the  wells,  respectively.  And  the  color  develops  in
opposite  to  the  amount  of VCAM-1/CD106  in  the  sample. The
color  development  is  stopped  and  the  intensity  of  the  color  is
measured.
DETECTION RANGE
The  standard  curve  concentrations  used  for  the  ELISA’s  were 
  3
8000 ng/ml, 4000 ng/ml, 2000 ng/ml, 1000 ng/ml, 500 ng/ml.
SPECIFICITY
This  assay  recognizes  VCAM-1/CD106.  No  significant
cross-reactivity or interference was observed.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standards （S1-S5）  5  
HRP-avidin
Conjugate
1 x 6 ml
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      1 x 6 ml
Standard  S1  S2  S3  S4  S5
Concentration（ng/ml）  500  1000  2000  4000  8000
STORAGE 
  4
1. Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt
and  the microtiter plate should be kept  in a sealed bag with
desiccants to minimize exposure to damp air. The test kit may
be used throughout the expiration date of the kit. Refer to the
package label for the expiration date.
2. Opened  test  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.
TECHNICAL HINTS
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 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.
3.  To  avoid  cross-contamination,  change  pipette  tips  between     
  5
additions of each standard  level, between sample additions,
and between reagent additions. Also, use separate reservoirs
for each reagent.
4. 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.
5.  Substrate Solution should remain colorless until added to the
plate. Keep Substrate Solution protected from light. Substrate
Solution should change from colorless to gradations of blue.
6.  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.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material. 
  6
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.
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 x g. Remove serum and assay immediay
or  aliquot  and  store  samples  at  -20°  C.  Avoid  repea ted
freeze-thaw cycles.
  Plasma    Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 x g within
30 minutes  of  collection.  Assay  immediay  or  aliquot  and
store samples at -20° C. Avoid repeated freeze-thaw  cycles.
Note: Grossly hemolyzed samples are not suitable for use in this assay. 
  7
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.
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 Conjugate to each well （not to Blank well）, Mix
well and then incubate for 1 hour at 37°C.  
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 HRP-avidin  to each well.  Incubate  for 30mins
at 37°C.
5.  Repeat the aspiration and wash five times as step 4.
6.  Add 50µl of Substrate A and 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. 
  8
7.  Add 50µl of Stop Solution to each well.  
8.  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 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 VCAM-1/CD106 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 
  9
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  Calibrator  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 Calibrator 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  
 

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