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CSB-E07967r 大鼠前列腺素E2(PGE2)ELISA Kit说明书

厦门慧嘉生物科技有限公司2013年5月17日 17:04 点击:880

 
Rat Prostaglandin E2 (PGE2)
ELISA Kit 
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  quantitative  determination  of  rat
PGE2 concentrations in tissue homogenates.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
 
  2
INTRODUCTION
One of  the prostaglandins, a group of hormone-like substances
that  participate  in  a wide  range  of  body  functions  such  as  the
contraction  and  relaxation  of  smooth  muscle,  the  dilation  and
constriction  of  blood  vessels,  control  of  blood  pressure,  and
modulation  of  inflammation.  Prostaglandin  E2  (PGE-2)  is
released  by  blood  vessel  walls  in  response  to  infection  or
inflammation that acts on the brain to induce fever. The enzyme
mPGES-1  is  involved  in  the  production  of  PGE2  and  is  an
important "switch" for activating the fever response.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
an  goat-anti-rabbit  antibody.  Standards  or  samples  are  then
added  to  the  appropriate  microtiter  plate  wells  with  a
HRP-conjugated  PGE2  and  antibody  preparation  specific  for
PGE2  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 
  3
nm.  The  concentration  of  PGE2  in  the  samples  is  then
determined  by  comparing  the  O.D.  of  the  samples  to  the
standard curve.
DETECTION RANGE
0.4 pg/ml-100 pg/ml. The standard curve concentrations used for
the ELISA’s were 100 pg/ml, 25 pg/ml, 6.25 pg/ml, 1.55 pg/ml,
0.4 pg/ml.
SPECIFICITY
This  assay  recognizes  recombinant  and  natural  rat  PGE2. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of rat PGE2 is typically less than
0.25 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. 
  4
MATERIALS PROVIDED
 
Standard  S 1  S 2  S 3  S 4  S 5
Concentration
(pg/ml)
0.4  1.55  6.25  25  100
 
  Reagent  Quantity  Storage
Unopened kit  Store at 2 - 8° C. Do not use beyond kit expiration  date.
Coated plate  1
May be stored for up to 1 month
at 2 - 8° C. Try to keep it in a
sealed aluminum foil bag,and
avoid the damp
Standards
(S1-S5)
5 x 0.5 ml  
Antibody    1 x 6 ml
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
 
Opened kit
Stop Solution  1 x 7 ml
  
May be stored for up to 1 month
at 2 - 8° C. 
  5
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  completely  dissolved.  Dilute  15  ml  of  Wash  Buffer
Concentrate into deionized or distilled water to prepare 300 ml
of Wash Buffer.
3.  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.
4. 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.
5.  To ensure accurate results, proper adhesion of plate sealers
during  incubation  steps  is  necessary.  Sealers  can  not  be
reused. 
  6
6.  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.
7.  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.
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
  Tissue  Homogenates  The  preparation  of  tissue 
  7
homogenates  will  vary  depending  upon  tissue  type.  Heart
and  lung  tissue  from  one  rat  was  rinsed  with  1X  PBS  to
remove excess blood, homogenized in 20 mL of 1X PBS and
stored overnight at -20° C. After two freeze-thaw c ycles were
performed  to  break  the  cell membranes,  the  homogenates
were  centrifuged  for  5 minutes  at  5000  x  g. The  supernate
was assayed and removed immediately. Alternatively, 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. Standards need test in duplicate.  
2.  Add 50µl of HRP-conjugate to each well (not to Blank well),
then  add  50µl  Antibody  to  each  well.  Mix  well  and  then
incubate for 1 hour at 37°C.   
  8
3.  Fill  each well with Wash Buffer  (about  200µl),  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
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,  Blank,  and
sample  and  subtract  the  optical  density  of  Blank.  Create  a
standard  curve  by  reducing  the  data  using  computer  software 
  9
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 PGE2 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  Calibrator  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 Calibrator Diluent and
repeat the assay. 
  10
  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.
PRECISION
Intra-assay Precision (Precision within an assay)
One sample whose concentration between  the highest and  the
second  standard  were  tested  twenty  times  on  one  plate  to
assess.
CV%<8%
Inter-assay Precision (Precision between assays)
One sample whose concentration between  the highest and  the
second  standard  were  tested  in  thirty-five  separate  assays  to
assess
CV%<10% 
  11
RECOVERY
The  recovery  of  PGF2  spiked  to  three  levels  throughout  the
range  of  the  assay  in  the  tissue  homogenates  and  cell  culture
supernates samples was evaluated.
 
Sample Type  Average %  Recovery Range
Rat Tissue
Homogenates(n
=6)
90  84 - 102%
 
Samples were spiked  then diluted prior  to assay as directed  in
Sample Preparation.
PLATE LAYOUT
Use  this  plate  layout  as  a  record  of  standards  and  samples
assayed. 
  
 

(来源: 厦门慧嘉生物科技有限公司


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