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Human Insulin （INS）
ELISA Kit
 
 
Catalog No. CSB-E05069h
（96T）
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  quantitative  determination  of  human  INS
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.
 
 
 
 
CUSABIO BIOTECH CO., LTD.
http://www.cusabio.com/    http://www.cusabio.cn/
: cusabio@cusabio.com    cusabio@cusabio.cn 
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INTRODUCTION
Insulin is a peptide hormone composed of 51 amino acid residues and has
a molecular weight of 5808 Da. It is produced in the islets of Langerhans
in  the  pancreas.  Insulin's  structure  varies  slightly  between  species  of
animal.  Insulin  has  extensive  effects  on  both  metabolism  and  several
other body systems （eg, vascular compliance）. Insulin causes most of the
body's cells  to  take up glucose  from  the blood  （including  liver, muscle,
and  fat  tissue cells）,  storing  it as glycogen  in  the  liver and muscle, and
stops  use  of  fat  as  an  energy  source. When  insulin  is  absent  （or  low）,
glucose is not taken up by most body cells and the body begins to use fat
as an energy source （ie, transfer of lipids from adipose tissue to the liver
for mobilization as an energy source）. As  its level  is a central metabolic
control mechanism, its status is also used as a control signal to other body
systems  （such as amino acid uptake by body cells）.  It has  several other
anabolic effects throughout the body.
PRINCIPLE OF THE ASSAY
The  microtiter  plate  provided  in  this  kit  has  been  pre-coated  with  an
antibody  specific  to  INS.  Standards  or  samples  are  then  added  to  the
appropriate  microtiter  plate  wells  with  a  HRP-conjugated  monoclonal
antibody  preparation  specific  for  INS  and  incubated.  Then  substrate
solution  is  added  to  each well. Only  those wells  that  contain  INS  and
HRP-conjugated  antibody  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 INS in the samples
is then determined by comparing the O.D. of the samples to the standard
curve. 
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DETECTION RANGE
8  µIU/ml-140  µIU/ml.  The  standard  curve  concentrations  used  for  the
ELISA’s were 140 µIU/ml, 80 µIU/ml, 32 µIU/ml, 16 µIU/ml, 8 µIU/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  human  INS.  No
significant cross-reactivity or interference was observed.
MATERIALS PROVIDED
Reagent      Quantity
Assay plate  1
Standards （S1-S5）  5  
HRP-conjugate  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
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. 
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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.  Standard    Reconstitute  the  Standards  with  0.5  ml  of  ddH2O,
respectively. Allow  the  standard  to  sit  for a minimum of 15 minutes
with gentle agitation prior to use.  
4.  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.
5.  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.
6.  To  ensure  accurate  results,  proper  adhesion  of  plate  sealers  during
incubation steps is necessary. Sealers can not be reused.
7.  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.
8.  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.
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
  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 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 
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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 HRP-conjugate  to each well  （not  to Blank well）. Mix
well and then incubate for 2 hour at 37°C.  
3.  Fill  each well with Wash Buffer  （about 350µl）,  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 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
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  INS
concentrations  versus  the  log  of  the  O.D.  and  the  best  fit  line  can  be 
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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.
  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.

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