Human Activin A（ACV-A） ELISA Kit
Catalog Number. CSB-E04486h
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.
If You Have Problems
Technical Service Contact information 
 
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.
 
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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.
 
 
 
 
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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.
 
 
 
 
 
 
 
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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  immediay  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  immediay  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  immediay. 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.
 
 
 
 
 
 
 
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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  compley
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
 
 
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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.
 
 
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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
 
 
 
 
 
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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. 
 

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