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Human influenza viruses IgG
ELISA Kit
 
 
Catalog No. CSB-E05007h    
（96 tests）
 
 
 
 
  This  immunoassay  kit  allows  for  the  in  vitro  semi-quantitative  determination  of
human influenza viruses IgG antibody concentrations in serum, plasma.
  Expiration date      six months from the date of manufacture
  FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
 
 
 
 
CUSABIO BIOTECH CO., LTD.
http://www.cusabio.com          http://www.cusabio.cn  
:    cusabio        info@cusabio.com     
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PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with
purified  human  influenza  viruses  antigen.  Samples  are  then
added  to  the  appropriate microtiter  plate  wells  and  incubated.
Then  add  Horseradish  Peroxidase  （HRP）-conjugated
anti-human IgG to each well and incubate. Finally, the substrate
solution is added to each well. The enzyme-substrate reaction is
terminated by the addition of stop solution and the color change
is measured spectrophotometrically at a wavelength of 450 nm ±
2  nm.  Calculate  the  valence  of  human  influenza  viruses  IgG
antibody in the samples.
SPECIFICITY
This assay recognizes human influenza viruses IgG antibody. No
significant cross-reactivity or interference was observed. 
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MATERIALS PROVIDED
Reagent  Quantity
Assay plate  1
Sample Diluent  1 x 20 ml
HRP-anti-human IgG Diluent      1 x 10 ml
HRP- anti-human IgG  1 x 120µl
Negative control  1 x 1 ml
Positive control  1 x 1ml
Wash Buffer
1 x 20 ml
（25×concentrate）
Substrate A  1 x 5ml
Substrate B  1 x 5 ml
Stop Solution  1 x 5ml
STORAGE
1. Unopened  test  kits  should  be  stored  at  2-8°C  upon  receipt
and  the microtiter plate should be kept  in a sealed bag. The
test kit may be used throughout the expiration date of the kit.
Refer to the package label for the expiration date. 
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2. Opened  test  kits  will  remain  stable  until  the  expiring  date
shown, provided it is stored as prescribed above.    
3.  A microtiter  plate  reader with  a  bandwidth  of  10  nm  or  less
and an optical density  range of 0-3 OD or greater at 450nm
wavelength  is  acceptable  for  use  in  absorbance
measurement.
TECHNICAL HINTS
  Bring all reagents and plate to room temperature for at least
30 minutes before use. Unused wells need store at 2-8℃and
avoid sunlight.
  Wash  Buffer    If  crystals  have  formed  in  the  concentrate,
warm  to  room  temperature and mix gently until  the crystals
have  compley  dissolved.  Dilute  20  ml  of  Wash  Buffer
Concentrate  into deionized or distilled water  to prepare 500
ml of Wash Buffer.
  Sample  Dilute  1µl  sample  using  200µl  Sample  Diluent
（1:201）, respectively.
  HRP-anti-human  IgG    Dilute  to  the  working  concentration
using HRP-anti-human IgG Diluent（1:100）, respectively. 
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  To  avoid  cross-contamination,  change  pipette  tips  between
sample additions, and between  reagent additions. Also, use
separate reservoirs for each reagent.
  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.
  Substrate Solution should remain colorless until added to the
plate. Keep Substrate Solution protected from light. Substrate
Solution should change from colorless to gradations of blue.
  Stop Solution should be added to the plate in the same order
as  the Substrate Solution. 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 Substrate Solution.
Precaution: The Stop Solution provided with this kit is an acid solution. Wear
eye, hand, face, and clothing protection when using this material. 
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OTHER SUPPLIES REQUIRED
  Microplate  reader capable of measuring absorbance at 450
nm, with the correction wavelength set at 540 nm or 570 nm.
  Pipettes and pipette tips.
  Deionized or distilled water.
  Squirt bottle, manifold dispenser, or automated microplate
washer.
SAMPLE COLLECTION AND STORAGE
  Serum    Use  a  serum  separator  tube  （SST）  and  allow
samples  to  clot  for  30 minutes  before  centrifugation  for  15
minutes at 1000 g. Remove serum and assay immediay or
aliquot  and  store  samples  at  -20°  C.  Avoid  repeated 
freeze-thaw cycles.
  Plasma    Collect plasma using citrate, EDTA, or heparin as
an anticoagulant. Centrifuge for 15 minutes at 1000 g within
30 minutes  of  collection.  Assay  immediay  or  aliquot  and
store samples at -20° C. Avoid repeated freeze-thaw  cycles.  
Note: Grossly hemolyzed samples are not suitable for use in this assay. 
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ASSAY PROCEDURE
Bring  all  reagents  and  samples  to  room  temperature  before  use.  It  is
recommended that all samples, and controls be assayed in duplicate.
1.  Add  100µl  negative  control,  positive  control  and Sample  to
each Sample well. Mix well, then  incubate for 30 minutes at
37°C.  
2.  Aspirate each well and wash, repeating the process for a total
of  five washes. Wash by  filling each well with Wash Buffer
（200µl） using a squirt bottle, multi-channel pipette, manifold
dispenser or autowasher. Complete removal of liquid at each
step  is  essential  to  good  performance. After  the  last wash,
remove  any  remaining  Wash  Buffer  by  aspirating  or
decanting.  Invert  the  plate  and  blot  it  against  clean  paper
towels.
3.  Add  100µl  of  HRP-conjugate  to  each  well.  Incubate  for
30minutes  at  37°C.  HRP-conjugate  may  appear  cloudy. 
Warm up  to  room  temperature and mix gently until solution
appears uniform.
4.  Wash plate five times as before. 
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5.  Add 50µl of Substrate A and Substrate B to each well, mix
well. Incubate for 10 minutes at 37°C. Keeping the  plate away
from drafts and other temperature fluctuations in the dark.
6.  Add 50µl of Stop Solution to each well.  
7.  Determine the optical density of each well within 10 minutes,
using a microplate reader set to 450 nm.
CALCULATION OF RESULTS
For  calculation  the  valence  of  human  influenza  viruses  IgG
antibody, compare the sample well with control.  
While ODsample≥2.1x ODnegative: Positive  
While ODsample＜2.1x ODnegative: Negative
LIMITATIONS OF THE PROCEDURE
  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. 
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  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
Quantikine  Immunoassay,  the  possibility  of  interference
cannot be excluded.

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