Human gonadotropin-releasing
hormone（GnRH） ELISA Kit
 
 
Catalog No. CSB-E06880h
（96T）
 
 
 
 
 
 
  This immunoassay kit allows for the in vitro quantitative determination of human
GnRH 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
Gonadotropin-releasing  hormone  （GnRH）,  also  known  as
Luteinizing-hormone releasing hormone （LHRH） and luliberin, is
a tropic peptide hormone responsible for the release of FSH and
LH from the anterior pituitary. GnRH is synthesized and released
from neurons within the hypothalamus.
The  gene,  GNRH1,  for  the  GNRH  precursor  is  located  on
chromosome 8. In mammals, the linear decapeptide end product
is  synthesized  from  a  92  amino  acid  preprohormone  in  the
preoptic anterior hypothalamus.
GnRH is considered a neurohormone, a hormone produced in a
specific neural cell and released at its neural terminal. A key area
for production of GNRH is the preoptic area of the hypothalamus,
that  contains  most  of  the  GNRH-secreting  neurons.  GnRH
neurons originate  in  the nose and migrate  into  the brain where
they  are  scattered  throughout  the  medial  septum  and
hypothalamus and connected by very long >1 mm long dendrites.
These bundle  together so  they receive shared synaptic  input, a
process that allows them to synchronize their GnRH release.
GnRH  is secreted  in  the hypophysial portal bloodstream at  the 
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median  eminence.  The  portal  blood  carries  the  GnRH  to  the
pituitary  gland,  which  contains  the  gonadotrope  cells,  where
GnRH  activates  its  own  receptor,  gonadotropin-releasing
hormone receptor （GNRHR）, a seven transmembrane G-protein
coupled  receptor  that  stimulates  the  beta  isoform  of
Phosphoinositide  phospholipase C, which  goes  on  to mobilize
calcium  and  protein  kinase C.  This  results  in  the  activation  of
proteins  involved  in  the  synthesis  and  secretion  of  the
gonadotropins, LH and FSH. GnRH  is degraded by proteolysis
within a few minutes.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an  antibody  specific  to GnRH, Standards  or  samples  are  then
added  to  the  appropriate  microtiter  plate  wells  with  a
biotin-conjugated  antibody  preparation  specific  for  GnRH  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 GnRH, biotin-conjugated antibody
and enzyme-conjugated Avidin will exhibit a change in color. The 
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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  GnRH  in  the  samples  is  then  determined  by
comparing the O.D. of the samples to the standard curve.
DETECTION RANGE
1.56 pg/ml-100 pg/ml. The standard curve concentrations used
for the ELISA’s were 100    pg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/ml,
6.25 pg/ml, 3.12 pg/ml，1.56 pg/ml.
SPECIFICITY
This assay  recognizes  recombinant and natural human GnRH.
No significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human GnRH  is  typically  less
than 0.5pg/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. 
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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. 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.
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    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 100
pg/ml. Allow the standard to sit for a minimum of 15 minutes
with  gentle  agitation  prior  to  making  serial  dilutions.  The 
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undiluted standard serves as  the high standard （100 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.
3.  Biotin-antibody    Centrifuge  the vial before opening. Dilute
to  the  working  concentration  using  Biotin-antibody
Diluent（1:100）, respectively.
4.  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.
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. 
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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
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 100µl of Standard, Blank, or Sample per well. Cover with 
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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: 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.
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 five times as step 4.
7.  Add 90µl of TMB Substrate to each well. Incubate for 10-30
minutes  at  37°C.  Keeping  the  plate  away  from  drafts   and 
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other temperature fluctuations in the dark.  
8.  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.
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.
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 GnRH concentrations versus  the  log of  the O.D.
and  the best  fit  line can be determined by  regression analysis. 
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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
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 
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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.
  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.