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廈門慧嘉生物科技有限公司
廈門慧嘉生物科技有限公司
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閱讀：283發(fā)布時間：2013-4-19
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.
: cusabio@ info@
2
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.
3
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.
6
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.
7
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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