Human Connective Tissue
Growth Factor （CTGF）
  Elisa Kit
 
 
 
Catalog No. CSB-E07875h
（96 tests）
 
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of human
CTGF 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.
 
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INTRODUCTION
CTGF  （connective  tissue  growth  factor）  is  a  cysteine-rich,
matrix-associated,  heparin-binding  protein.  In  vitro,  CTGF
mirrors some of the effects of TGF beta on skin fibroblasts, such
as  stimulation  of  extracellular  matrix  production,  chemotaxis,
proliferation  and  integrin  expression.  CTGF  can  promote
endothelial cell growth, migration, adhesion and survival and  is
thus  implicated  in  endothelial  cell  function  and  angiogenesis.
CTGF  gene  expression  had  been  demonstrated  in  human
trabecular  meshwork  cells,  ciliary  body,  retinal  vascular
endothelial  cells,  proliferative  vitreoretinopathy  membranes,
choroidal neovascular membranes, cataractous plaque, corneal
scars, tear fluid, and pterygia.
CTGF  binds  to  perlecan,  a  proteoglycan  which  has  been
localised  in  synovium,  cartilage  and  numerous  other  tissues.
CTGF has been implicated in extracellular matrix remodelling in
wound healing, scleroderma and other fibrotic processes, as it is
capable of upregulating both matrix metalloproteinases （MMPs）
and  their  inhibitors  （TIMPs）. Therefore, CTGF has  the potential
to  activate  both  the  synthesis  and  degradation  of  the
extracellular matrix. 
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an  antibody  specific  to CTGF. Standards  or  samples  are  then
added to the appropriate microtiter plate wells with a Horseradish
Peroxidase  （HRP） -conjugated antibody preparation specific  for
CTGF  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  CTGF  in  the  samples  is  then
determined  by  comparing  the  O.D.  of  the  samples  to  the
standard curve.
DETECTION RANGE
3.75 pg/ml-120 pg/ml. The standard curve concentrations used
for  the  ELISA’s were  120  pg/ml,  60  pg/ml,  26.25  pg/ml,  11.25
pg/ml, 3.75 pg/ml.
SPECIFICITY
This  assay  recognizes  human  CTGF.  No  significant
cross-reactivity or interference was observed. 
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SENSITIVITY
The minimum detectable dose of human CTGF  is  typically  less
than 1.87 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  5 x 0.5 ml
HRP-conjugate  1 x 6 ml
Substrate A  1 x 7 ml
Substrate B  1 x 7 ml
Stop Solution      1 x 7 ml
Standard  S1  S2  S3  S4  S5
Concentration
（pg/ml）
3.75  11.25  26.25  60  120
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. 
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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.
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 
  6
minutes at 1000 x g. Remove serum and assay immediay
or aliquot and store samples at -20°C. Centrifuge t he 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 x 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.  
2.  Add  50µl  of  HRP-Conjugate  to  each  well  （Not  to  Blank!）.
Incubate for 1 hour at 37°C. 
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3.  Aspirate  each  well  and  wash,  repeating  the  process  three
times  for a  total of  three washes. Wash by  filling each well
with  ddH2O  （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
solution 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.
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 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.
6.  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 CTGF  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 and repeat the assay. 
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  Any  variation  in  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. 
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  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.