Human plasminogen activator inhibitor  
（PAI） ELISA Kit 
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  quantitative  determination  of  human PAI
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. 
 
 
 
INTRODUCTION
Plasminogen  activator  inhibitor  （PAI）  is  serpin  family  member  that  inhibits  tissue-  and
urokinase-type  plaminogen  activators  （t-PA,  u-PA）. This  protein  appears  to  be  an  important
regulator  of  plasminogen  activation  by  t-PA  and  extracellular  proteolysis  by  u-PA.  The
plasminogen activator proteolytic enzyme  systems are  important not only  for  fibrinolysis but
also for extracellular matrix remodeling, and have been implicated in a number of normal and
pathological processes  including  angiogenesis, ovulation  and  embryogenesis,  thrombotic  and
hemorrhagic disorders, connective tissue diseases, neoplasm and sepsis.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an antibody specific to PAI.
Standards  or  samples  are  then  added  to  the  appropriate  microtiter  plate  wells  with  a
biotin-conjugated polyclonal  antibody preparation  specific  for PAI  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 PAI, 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 PAI  in  the samples  is  then determined by comparing  the O.D. of
the samples to the standard curve.
DETECTION RANGE
1.56 ng/ml-100 ng/ml. The standard curve concentrations used for the ELISA’s were 100ng/ml,
50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml, 1.56 ng/ml.
SPECIFICITY
This assay  recognizes  recombinant and natural human PAI. No  significant cross-reactivity or
interference was observed.
SENSITIVITY
The minimum detectable dose of human PAI is typically less than 0.39 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
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 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.
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    Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution
produces  a  stock  solution  of  100  ng/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 ng/ml）. The Sample Diluent serves as  the zero standard
（0 ng/ml）.
3.  Biotin-antibody    Dilute  to  the working  concentration  specified  on  the  vial  label  using
Biotin-antibody Diluent（1:100）, respectively.
4.  HRP-avidin    Dilute  to  the  working  concentration  specified  on  the  vial  label  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.
  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt bottle, manifold dispenser, or automated microplate washer.
SAMPLE COLLECTION AND STORAGE
  Cell Culture Supernates    Remove particulates by centrifugation and assay immediay
or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles.
  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. Avoid repeated 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.
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.  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 by  filling each well with Wash Buffer  （200µl） using a  squirt bottle, multi-channel
pipette,  manifold  dispenser  or  autowasher.  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.
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 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. 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
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 PAI 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 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
Quantikine Immunoassay, the possibility of interference cannot be excluded.
TECHNICAL HINTS
  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  until  added  to  the  plate.  Keep  Substrate
Solution  protected  from  light.  Substrate  Solution  should  change  from  colorless  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.