Human angiotensin converting enzyme 2 （ACE2）
ELISA Kit
Catalog Number. CSB-E04489h
For  the  quantitative  determination  of  human  angiotensin  converting
enzyme  2  （ACE2）  concentrations  in  serum,  plasma  and  cell  culture
supernates.
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 ACE2 has been pre-coated onto a microplate. Standards
and samples are pipetted into the wells and any ACE2 present is bound by the
immobilized  antibody.  After  removing  any  unbound  substances,  a
biotin-conjugated antibody specific for ACE2 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
ACE2  bound  in  the  initial  step.  The  color  development  is  stopped  and  the
intensity of the color is measured.
DETECTION RANGE
0.156 ng/ml-10 ng/ml.
SENSITIVITY
The minimum detectable dose of human ACE2 is typically less than 0.039 ng/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
ACE2. No significant cross-reactivity or interference between human ACE2 and
analogues was observed.
Note: Limited by current skills and knowledge, it is impossible for us to complete
the  cross-reactivity  detection  between  human  ACE2  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 the kit beyond the 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  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.
?  Cell Culture Supernates    Remove particulates by centrifugation  for 15
minutes at 1000 x g, 2 - 8°C and assay  immediay 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 compley 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  compley
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
10 ng/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 （10 ng/ml）. Sample Diluent serves as the zero standard （0
ng/ml）.
 
 
 
 
 
 
 
 
 
Tube  S7  S6  S5  S4  S3  S2  S1  S0
ng/ml  10  5  2.5  1.25  0.625  0.312  0.156  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 ACE2 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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