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现在位置不用本金就能赚钱的方法>技术资料不用本金就能赚钱的方法>产品信息>说明书>CSB-E04496h_人角化细胞内分泌因子(KAF)双调蛋白(AR)ELISA试剂盒Human_keratinocyte_autocrine_factor_Amphiregulin,KAF_AR_ELISA_Kit

CSB-E04496h_人角化细胞内分泌因子(KAF)双调蛋白(AR)ELISA试剂盒Human_keratinocyte_autocrine_factor_Amphiregulin,KAF_AR_ELISA_Kit

厦门慧嘉生物科技有限公司2013年5月23日 15:31 点击:697

 

 
 人角化细胞内分泌因子(KAF)双调蛋白(AR)ELISA试剂盒
Human keratinocyte autocrine factor/amphiregulin 
(KAF/AR) ELISA Kit 
 
For  the  quantitative  determination  of  human  keratinocyte  autocrine 
factor/amphiregulin (KAF/AR) concentrations in serum, plasma. 
This package insert must be read in its entirety before using this product. 
 
 
In order to obtain higher efficiency service, please ready to supply the lot number 
of the kit to us (found on the outside of the box).   2 
PRINCIPLE OF THE ASSAY 
This assay employs the quantitative sandwich enzyme immunoassay technique. 
Antibody specific for KAF/AR has been pre-coated onto a microplate. Standards 
and samples are pipetted into the wells and any KAF/AR present is bound by the 
immobilized  antibody.  After  removing  any  unbound  substances,  a 
biotin-conjugated  antibody  specific  for  KAF/AR  is  added  to  the  wells.  After 
washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. 
Following a wash  to  remove any unbound avidin-enzyme  reagent, a substrate 
solution is added to the wells and color develops in proportion to the amount of 
KAF/AR  bound  in  the  initial  step.  The  color  development  is  stopped  and  the 
intensity of the color is measured. 
DETECTION RANGE 
4.7 pg/ml-300 pg/ml. 
SENSITIVITY 
The minimum detectable dose of human KAF/AR is typically less than 1.2 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.  It was 
determined the mean O.D value of 20 replicates of the zero standard added by 
their three standard deviations. 
SPECIFICITY 
This assay has high sensitivity and excellent specificity  for detection of human 
KAF/AR. No significant cross-reactivity or interference between human KAF/AR 
and analogues was observed. 
Note: Limited by current skills and knowledge, it is impossible for us to complete 
the  cross-reactivity  detection  between  human  KAF/AR  and  all  the  analogues, 
therefore, cross reaction may still exist.   3 
PRECISION   
Intra-assay Precision (Precision within an assay): CV%<8% 
Three samples of known concentration were tested twenty times on one plate to 
assess.   
Inter-assay Precision (Precision between assays): CV%<10% 
Three samples of known concentration were tested in twenty assays to assess.   
LIMITATIONS OF THE PROCEDURE 
?  FOR RESEARCH USE ONLY. NOT FOR USE  IN DIAGNOSTIC 
PROCEDURES. 
?  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 Sample Diluent and repeat the assay. 
?  Any  variation  in  Sample 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  Immunoassay,  the  possibility  of 
interference cannot be excluded. 
 
 
 
 
   4 
MATERIALS PROVIDED 
Reagents  Quantity 
Assay plate (12 x 8 coated Microwells)  1(96 wells) 
Standard (Freeze dried)  2 
Biotin-antibody (100 x concentrate)    1 x 120 μl 
HRP-avidin (100 x concentrate)  1 x 120 μl 
Biotin-antibody Diluent      1 x 10 ml 
HRP-avidin Diluent        1 x 10 ml 
Sample Diluent      1 x 20 ml 
Wash Buffer (25 x concentrate)  1 x 20 ml 
TMB Substrate    1 x 10 ml 
Stop Solution      1 x 10 ml 
Adhesive Strip (For 96 wells)  4 
Instruction manual  1 
STORAGE 
Unopened kit  Store at 2 - 8°C. Do not use past kit expiration date 
Opened kit 
Coated assay 
plate 
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. 
Standard  May be stored for up to 1 month at 2 - 8° C. If 
don’t make recent use, better keep it store at 
-20°C. 
Biotin-antibody   
HRP-avidin 
Biotin-antibody 
Diluent 
May be stored for up to 1 month at 2 - 8°C. 
HRP-avidin 
Diluent 
Sample 
Diluent 
Wash Buffer 
TMB 
Substrate 
Stop Solution 
*Provided this is within the expiration date of the kit.   5 
OTHER SUPPLIES REQUIRED 
?  Microplate reader capable of measuring absorbance at 450 nm, with  the 
correction wavelength set at 540 nm or 570 nm. 
?  An  incubator  which  can  provide  stable  incubation  conditions  up  to 
37°C±0.5°C. 
?  Squirt bottle, manifold dispenser, or automated microplate washer. 
?  Absorbent paper for blotting the microtiter plate. 
?  100 mL and 500 mL graduated cylinders. 
?  Deionized or distilled water. 
?  Pipettes and pipette tips. 
?  Test tubes for dilution. 
PRECAUTIONS 
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, 
and clothing protection when using this material. 
 
 
 
 
 
 
   6 
SAMPLE COLLECTION AND STORAGE 
?  Serum    Use a serum separator tube (SST) and allow samples to clot for 
two hours at  room  temperature or overnight at 4°C before centrifugation 
for  15  minutes  at  1000  ×g.  Remove  serum  and  assay  immediately  or 
aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw 
cycles. 
?  Plasma    Collect  plasma  using  EDTA,  or  heparin  as  an  anticoagulant. 
Centrifuge  for  15  minutes  at  1000  ×g  at  2-8°C  within  30  minutes  of 
collection.  Assay  immediately  or  aliquot  and  store  samples  at  -20°C  or 
-80°C. Avoid repeated freeze-thaw cycles. 
 
 
 
 
 
 
 
 
   7 
Note: 
1.  CUSABIO  is  only  responsible  for  the  kit  itself,  but  not  for  the  samples 
consumed  during  the  assay.  The  user  should  calculate  the  possible 
amount of  the samples used  in  the whole  test. Please  reserve sufficient 
samples in advance. 
2.  Samples  to  be  used  within  5  days may  be  stored  at  2-8°C,  otherwise 
samples must be stored at -20°C (≤1month) or -80°C (≤2month) to avoid 
loss of bioactivity and contamination. 
3.  Grossly hemolyzed samples are not suitable for use in this assay. 
4.  If the samples are not indicated in the manual, a preliminary experiment to 
determine the validity of the kit is necessary.   
5.  Please predict  the concentration before assaying.  If values  for  these are 
not  within  the  range  of  the  standard  curve,  users  must  determine  the 
optimal sample dilutions for their particular experiments. 
6.  Tissue or cell extraction samples prepared by chemical  lysis buffer may 
cause unexpected ELISA results due to the impacts of certain chemicals. 
7.  Owing  to  the  possibility  of  mismatching  between  antigen  from  other 
resource  and  antibody  used  in  our  kits  (e.g.,  antibody  targets 
conformational  epitope  rather  than  linear  epitope),  some  native  or 
recombinant proteins from other manufacturers may not be recognized by 
our products. 
8.  Influenced  by  the  factors  including  cell  viability,  cell  number  and  also 
sampling time, samples from cell culture supernatant may not be detected 
by the kit. 
9.  Fresh samples without  long  time storage are  recommended  for  the  test. 
Otherwise,  protein degradation and denaturalization may occur  in  those 
samples and finally lead to wrong results. 
 
 
 
   8 
REAGENT PREPARATION 
Note:   
?  Kindly use graduated containers to prepare the reagent. Please don’t 
prepare the reagent directly in the Diluent vials provided in the kit. 
?  Bring all reagents to room temperature (18-25°C) before use for 30min. 
?  Prepare  fresh  standard  for each assay. Use within 4 hours and discard 
after use. 
?  Making serial dilution in the wells directly is not permitted.   
?  Please carefully  reconstitute Standards according  to  the  instruction, and 
avoid foaming and mix gently until the crystals have completely dissolved. 
To  minimize  imprecision  caused  by  pipetting,  use  small  volumes  and 
ensure that pipettors are calibrated. It is recommended to suck more than 
10μl for once pipetting. 
?  Distilled water  is  recommended  to be  used  to make  the preparation  for 
reagents  or  samples.  Contaminated  water  or  container  for  reagent 
preparation will influence the detection result. 
1.  Biotin-antibody (1x) - Centrifuge the vial before opening.   
Biotin-antibody requires a 100-fold dilution. A suggested 100-fold dilution 
is 10 μl of Biotin-antibody + 990 μl of Biotin-antibody Diluent. 
2.  HRP-avidin (1x) - Centrifuge the vial before opening.   
HRP-avidin requires a 100-fold dilution. A suggested 100-fold dilution is 10 
μl of HRP-avidin + 990 μl of HRP-avidin Diluent. 
3.  Wash Buffer(1x)-  If crystals have  formed  in  the concentrate, warm up  to    
room  temperature  and  mix  gently  until  the  crystals  have  completely 
dissolved. Dilute 20 ml of Wash Buffer Concentrate (25 x) into deionized or 
distilled water to prepare 500 ml of Wash Buffer (1 x). 
 
   9 
4.  Standard   
Centrifuge the standard vial at 6000-10000rpm for 30s.   
Reconstitute  the  Standard  with  1.0  ml  of  Sample  Diluent.  Do  not 
substitute other diluents. This reconstitution produces a stock solution of 
300 pg/ml. Mix  the standard  to ensure complete  reconstitution and allow 
the standard to sit for a minimum of 15 minutes with gentle agitation prior 
to making dilutions.   
 
Pipette 250 μl of Sample Diluent  into each  tube  (S0-S6). Use  the stock 
solution  to  produce  a  2-fold  dilution  series  (below).  Mix  each  tube 
thoroughly before the next transfer. The undiluted Standard serves as the 
high standard (300 pg/ml). Sample Diluent serves as the zero standard (0 
pg/ml). 
 
 
 
 
 
 
 
 
 
Tube  S7  S6  S5  S4  S3  S2  S1  S0 
pg/ml  300  150  75  37.5  18.8  9.4  4.7  0 
 
 
 
 
 
 
   10 
ASSAY PROCEDURE 
Bring all reagents and samples to room temperature before use. Centrifuge 
the sample again after thawing before the assay. It is recommended that all 
samples and standards be assayed in duplicate.   
1.  Prepare all  reagents, working standards, and samples as directed  in  the 
previous sections. 
2.  Refer  to  the Assay Layout Sheet  to determine  the number of wells  to be 
used and put any remaining wells and  the desiccant back into  the pouch 
and seal the ziploc, store unused wells at 4°C. 
3.  Add 100μl of standard and sample per well. Cover with the adhesive strip 
provided. Incubate for 2 hours at 37°C. A plate layout is provided to record 
standards and samples assayed. 
4.  Remove the liquid of each well, don’t wash.   
5.  Add  100μl  of  Biotin-antibody  (1x)  to  each  well.  Cover  with  a  new 
adhesive  strip.  Incubate  for  1  hour  at  37°C.  (Biotin-antibody  (1x) may 
appear cloudy. Warm up to room temperature and mix gently until solution 
appears uniform.) 
6.  Aspirate each well and wash, repeating the process two times for a total of 
three washes. Wash by filling each well with Wash Buffer (200μl) using a 
squirt  bottle,  multi-channel  pipette,  manifold  dispenser,  or  autowasher, 
and  let  it stand  for 2 minutes, complete  removal of  liquid at each step  is 
essential to good performance. After the last wash, remove any remaining 
wash Buffer by aspirating ordecanting. Invert the plate and blot it against 
clean paper towels. 
7.  Add 100μl of HRP-avidin (1x) to each well. Cover the microtiter plate with 
a new adhesive strip. Incubate for 1 hour at 37°C. 
8.  Repeat the aspiration/wash process for five times as in step 6. 
9.  Add 90μl of TMB Substrate  to each well.  Incubate  for 15-30 minutes at 
37°C. Protect from light. 
10.  Add  50μl  of Stop Solution  to  each well,  gently  tap  the  plate  to ensure 
thorough mixing.   11 
11.  Determine  the  optical  density  of  each  well  within  5  minutes,  using  a 
microplate reader set to 450 nm. If wavelength correction is available, set 
to 540 nm or 570 nm. Subtract  readings at 540 nm or 570 nm  from  the 
readings at 450 nm. This subtraction will correct  for optical imperfections 
in the plate. Readings made directly at 450 nm without correction may be 
higher and less accurate.   
*Samples may require dilution. Please refer to Sample Preparation section. 
Note: 
1.  The  final  experimental  results  will  be  closely  related  to  validity  of  the 
products,  operation  skills  of  the  end  users  and  the  experimental 
environments.   
2.  Samples or reagents addition: Please use the freshly prepared Standard. 
Please carefully add samples to wells and mix gently to avoid foaming. Do 
not  touch  the well wall as possible. For each step  in  the procedure,  total 
dispensing  time  for  addition  of  reagents  or  samples  to  the  assay  plate 
should  not  exceed  10 minutes.  This will  ensure  equal  elapsed  time  for 
each pipetting step, without  interruption. Duplication of all standards and 
specimens,  although  not  required,  is  recommended.  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. 
3.  Incubation: To ensure accurate  results, proper adhesion of plate sealers 
during incubation steps is necessary. Do not allow wells to sit uncovered 
for extended periods between incubation steps. Once reagents have been 
added to the well strips, DO NOT let the strips DRY at any time during the 
assay. Incubation time and temperature must be observed. 
4.  Washing:  The wash  procedure  is  critical. Complete  removal  of  liquid  at 
each step  is essential  to good performance. After  the  last wash,  remove 
any remaining Wash Solution by aspirating or decanting and remove any 
drop  of  water  and  fingerprint  on  the  bottom  of  the  plate.  Insufficient 
washing  will  result  in  poor  precision  and  falsely  elevated  absorbance 
reading. 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.   12 
5.  Controlling of reaction time: Observe the change of color after adding TMB 
Substrate  (e.g.  observation  once  every  10  minutes),  TMB  Substrate 
should change from colorless or light blue to gradations of blue. If the color 
is  too  deep,  add  Stop  Solution  in  advance  to  avoid  excessively  strong 
reaction which will result in inaccurate absorbance reading. 
6.  TMB  Substrate  is  easily  contaminated.  TMB  Substrate  should  remain 
colorless or light blue until added to the plate. Please protect it from light. 
7.  Stop Solution should be added to the plate in the same order as the TMB 
Substrate. 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 TMB Substrate. 
 
 
 
 
 
 
 
 
 
   13 
ASSAY PROCEDURE SUMMARY 
 
 
 
 
 
 
 
 
 
 
*Samples may require dilution. Please refer to Sample Preparation section. 
 
 
   14 
CALCULATION OF RESULTS 
Using the professional soft "Curve Expert 1.3" to make a standard curve is 
recommended, which can be downloaded from our web. 
Average  the duplicate readings  for each standard 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 x-axis against  the concentration on  the y-axis and draw a best  fit curve 
through the points on the graph. The data may be linearized by plotting the log of 
the KAF/AR 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. 
 
(来源: 厦门慧嘉生物科技有限公司


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