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Human Corticotropin Releasing
Hormone （CRH）
ELISA Kit
 
 
 
Catalog No. CSB-E06872h
（96T）
 
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of human
CRH concentrations in serum 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
Corticotropin-releasing  hormone  （CRH）  is  a  41-amino  acid
peptide derived  from a 191-amino acid preprohormone. CRH  is
secreted  by  the  paraventricular  nucleus  （PVN）  of  the
hypothalamus  in  response  to stress. Marked  reduction  in CRH
has been observed in association with Alzheimer's disease, and
autosomal  recessive  hypothalamic  corticotropin  deficiency  has
multiple and potentially-fatal metabolic  consequences  including
hypoglycemia and hepatitis. In addition to being produced in the
hypothalamus,  CRH  is  also  synthesized  in  peripheral  tissues,
such as T lymphocytes, and is highly expressed in the placenta.
In  the placenta, CRH  is a marker  that determines  the  length of
gestation  and  the  timing  of  parturition  and  delivery.  A  rapid
increase  in  circulating  levels  of  CRH  occurs  at  the  onset  of
parturition, suggesting that, in addition to its metabolic functions,
CRH may act as a trigger for parturition.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with 
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goat-anti-rabbit antibody. Standards or samples are  then added
to  the  appropriate  microtiter  plate  wells  with  a  Horseradish
Peroxidase  （HRP）-conjugated  CRH  and  antibody  preparation
specific  for CRH,  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  CRH  in  the  samples  is  then
determined  by  comparing  the  O.D.  of  the  samples  to  the
standard curve.
DETECTION RANGE
0.8 ng/ml-40 ng/ml. The standard curve concentrations used for
the  ELISA’s  were  40  ng/ml,  20  ng/ml,  8  ng/ml,  2.4  ng/ml,  0.8
ng/ml.
SPECIFICITY
This  assay  recognizes  human  CRH.  No  significant
cross-reactivity or interference was observed. 
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SENSITIVITY
The minimum  detectable  dose  of  human CRH  is  typically  less
than 1 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  5 x 0.5 ml
HRP-Conjugate  1 x 6ml
Antibody  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
 
Standard  S1  S2  S3  S4  S5
Concentration
（ng/ml）
0.8    2.4    8    20    40   
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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
1.  Bring all reagents to room temperature before use.  
2.  Wash  Buffer    If  crystals  have  formed  in  the  concentrate,
warm  up  to  room  temperature  and  mix  gently  until  the 
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crystals  have  compley  dissolved.  Dilute  15  ml  of Wash
Buffer Concentrate into deionized or distilled water to prepare
300 ml of Wash Buffer.
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.
  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 
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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 without  any  solution.  Add  50µl  of  Standard  or
Sample per well.  
2.  Add 50µl of HRP-Conjugate and 50µl of Antibody  to each
well. Not to Blank well!
3.  Cover with the adhesive strip. Incubate for 2 hours at 37° C.  
4.  Aspirate  each  well  and  wash,  repeating  the  process  three
times  for a  total of  three washes. Wash: Fill each well with
Wash  Buffer  （200µl）  and  let  it  stand  for  2  minutes,  then
remove  the  liquid  by  flicking  the  plate  over  a  sink.  The
remaining drops are removed by patting the plate on a paper
towel. Complete removal of liquid at each step is essential to
good performance. 
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5.  Add 50µl of Substrate A and 50µl of 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.
6.  Add 50µl of Stop Solution to each well. If color change does
not appear uniform, gently  tap  the plate  to ensure  thorough
mixing.
7.  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.
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 CRH concentrations versus the log of the O.D. and 
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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 with the appropriate Diluent and repeat the
assay.
  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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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.
  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. 
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  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.