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CSB-E04486h_人活化素A(ACV-A)ELISA试剂盒Human_Activin_A,ACV-A_ELISA_Kit

厦门慧嘉生物科技有限公司2013年5月23日 15:28 点击:1009

人活化素A(ACV-A)ELISA试剂盒

 
Human Activin A(ACV-A) ELISA Kit 
 
For the quantitative determination of human activin A (ACV-A) 
concentrations in serum, plasma, tissue homogenates. 
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 ACV-A has been pre-coated onto a microplate. Standards 
and samples are pipetted  into  the wells with a Horseradish Peroxidase  (HRP) 
conjugated  antibody  specific  for  ACV-A.  Following  a  wash  to  remove  any 
unbound reagent, a substrate solution is added to the wells and color develops in 
proportion  to  the  amount  of  ACV-A  bound  in  the  initial  step.  The  color 
development is stopped and the intensity of the color is measured. 
DETECTION RANGE 
66.7 pg/ml-2000 pg/ml. 
SENSITIVITY 
The minimum detectable dose of human ACV-A is typically less than 16.7 pg/ml. 
The sensitivity of  this assay, or Lower Limit of Detection  (LLD) was defined as 
the lowest human ACV-A 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 
ACV-A. No significant cross-reactivity or interference between human ACV-A and 
analogues was observed. 
Note: Limited by current skills and knowledge, it is impossible for us to complete 
the  cross-reactivity  detection  between  human  ACV-A  and  all  the  analogues, 
therefore, cross reaction may still exist. 
 
   3 
PRECISION   
Intra-assay Precision (Precision within an assay): CV%<15% 
Three samples of known concentration were tested twenty times on one plate to 
assess.   
Inter-assay Precision (Precision between assays): CV%<15% 
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 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  Immunoassay,  the  possibility  of 
interference cannot be excluded. 
 
 
 
 
   4 
MATERIALS PROVIDED 
Reagents  Quantity 
Assay plate  1(96 wells) 
Standard  6 (lyophilized) 
HRP-conjugate    1 x 6 ml 
Wash Buffer (20 x concentrate)  1 x 15 ml 
Substrate A  1 x 7 ml 
Substrate B  1 x 7 ml 
Stop Solution      1 x 7 ml 
Adhesive Strip (For 96 wells)  4 
Instruction manual  1 
STORAGE 
Unopened kit  Store at 2 - 8°C. Do not use the kit beyond the expiration date. 
Opened kit  May be stored for up to one month at 2 - 8° C. 
*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 600 nm - 630 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. 
?  Tissue  Homogenates    100mg  tissue  was  rinsed  with  1XPBS, 
homogenized in 1 ml of 1X PBS and stored overnight at -20°C. After two 
freeze-thaw  cycles  were  performed  to  break  the  cell  membranes,  the 
homogenates were  centrifuged  for 5 minutes at 5000  x g,  2  - 8°C. The 
supernate was  assayed  and  removed  immediately. Alternatively,  aliquot 
and store samples at  -20°C or  -80°C. Centrifuge  the sample again after 
thawing before the assay. 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.   
?  Bring all reagents to room temperature (18-25°C) before use for 30min. 
?  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、 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 15 ml of Wash Buffer Concentrate (20 x) into deionized or 
distilled water to prepare 300 ml of Wash Buffer (1 x). 
2、  Standard   
Centrifuge the standard vial at 6000-10000rpm for 30s.   
Reconstitute  each  lyophilized  Standard  with  0.5 ml  of  ddH2O. Mix  the 
standard  to ensure complete  reconstitution and allow  the standard  to sit 
for a minimum of 10 minutes with gentle agitation prior to use. 
Standard  S0  S1  S2  S3  S4  S5 
Concentration 
(pg/ml) 
0  66.7  166.7  500  1000  2000 
 
 
   9 
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.  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 50μl of Standard or Sample per well. Standard need test in duplicate.   
4.  Add 50μl of HRP-conjugate to each well. Mix well and then incubate for 1 
hour at 37°C.   
5.  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 10 seconds, 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. 
6.  Add 50μl of Substrate A and 50μl of 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. 
7.  Add  50μl  of Stop Solution  to  each well,  gently  tap  the  plate  to ensure 
thorough mixing.   
8.  Determine  the  optical  density  of  each  well  within  10  minutes,  using  a 
microplate reader set to 450 nm. 
 
 
   10 
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 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. 
5.  Controlling  of  reaction  time:  Observe  the  change  of  color  after  adding 
Substrates  (e.g. observation once every 10 minutes). Substrates  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.  Substrates are easily contaminated. Substrates 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 
Substrates. 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 Substrates.   11 
ASSAY PROCEDURE SUMMARY 
 
 
 
 
 
   12 
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 ACV-A 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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