Human Interleukin 18（IL-18）
ELISA Kit
 
 
Catalog No. CSB-E07450h
 
（96 T）
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of human
IL-18 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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INTRODUCTION
Interleukin-18 also known as IL18 is a protein which in humans is
encoded by the IL18 gene. The protein encoded by this gene is a
proinflammatory  cytokine.  IL-18  is  a  cytokine  produced  by
macrophages and other cells that belongs to the IL-1 superfamily.
IL-18 works together with IL-12 to induce cell-mediated immunity
following infection with microbial products like lipopolysaccharide
（LPS）. After stimulation with  IL-18, natural  killer  （NK） cells and
certain  T  cells  release  another  important  cytokine  called
interferon-γ  （IFN-γ） or  type  II  interferon  that plays an  important
role  in  activating  the  macrophages  or  other  cells.  The
combination of this cytokine and IL12 has been shown to inhibit
IL4  dependent  IgE  and  IgG1  production,  and  enhance  IgG2a
production  in  B  cells.  IL-18  binding  protein  （IL18BP）  can
specifically  interact  with  this  cytokine,  and  thus  negatively
regulate its biological activity.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an  antibody  specific  to  IL-18.  Standards  or  samples  are  then 
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added  to  the  appropriate  microtiter  plate  wells  with  a
biotin-conjugated  antibody  preparation  specific  for  IL-18  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  IL-18,  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  IL-18  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, 62.5 pg/ml, 31.2 pg/ml,15.6 pg/ml
SPECIFICITY
This assay recognizes recombinant and natural human IL-18. No
significant cross-reactivity or interference was observed. 
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SENSITIVITY
The minimum  detectable  dose  of  human  IL-18  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.
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 
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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
Bring all reagents to room temperature before use.  
  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 
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Buffer Concentrate into deionized or distilled water to prepare
500 ml of Wash Buffer.
  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.
  Biotin-antibody    Centrifuge  the vial before opening. Dilute
to  the  working  concentration  using  Biotin-antibody
Diluent（1:100）, respectively.
  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 
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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.  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 
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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 five 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.  
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. 
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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 IL-18 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 
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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
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, 
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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.