人血管緊張素Ⅱ（ANG-Ⅱ）ELISA試劑盒

Human Angiotensin Ⅱ （ANG-Ⅱ） ELISA Kit 

Catalog Number. CSB-E04493h 

For  the  quantitative  determination  of  human  angiotensin  Ⅱ  （ANG-Ⅱ） 

concentrations  in  serum,  plasma,  cell  culture  supernates,  tissue 

homogenates. 

: tech@cusabio.com   

Web:    www.cusabio.com   

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 ANG-Ⅱ has been pre-coated onto a microplate. Standards 

and samples are pipetted into the wells and any ANG-Ⅱ present is bound by the 

immobilized  antibody.  After  removing  any  unbound  substances,  a 

biotin-conjugated  antibody  specific  for  ANG-Ⅱ  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 

ANG-Ⅱ  bound  in  the  initial  step.  The  color  development  is  stopped  and  the 

intensity of the color is measured. 

DETECTION RANGE 

0.625 ng/ml-40 ng/ml. 

SENSITIVITY 

The minimum  detectable  dose  of  human  ANG-Ⅱ  is  typically  less  than  0.156 

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 

ANG-Ⅱ. No significant cross-reactivity or  interference between human ANG-Ⅱ 

and analogues was observed. 

Note: Limited by current skills and knowledge, it is impossible for us to complete 

the  cross-reactivity  detection  between  human  ANG-Ⅱ  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. 

?  100ml and 500ml 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. 

?  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. 

   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 

40 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 （40 ng/ml）. Sample Diluent serves as the zero standard （0 

ng/ml）. 

Tube  S7  S6  S5  S4  S3  S2  S1  S0 

ng/ml  40  20  10  5  2.5  1.25  0.625  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 ANG-Ⅱ 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.