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Avian Influenza
H5-HA(Ab)
ELISA KIT FOR DETECTION OF ANTIBODIES TO HEMAGGLUTININ (HA)
OF INFLUENZA A VIRUS, H5 STRAIN
Two-Step Incubation, ELISA Blocking Method
INSTRUCTIONS FOR USE
This kit is an enzyme-linked immunosorbent
assay (ELISA) for qualitative detection of antibodies
(anti-HA) to hemagglutinin(HA) of Influenza A viruses,
H5 strain in human/avian serum or plasma samples. It is
intended for clinical identification of infection with the
H5 strain of type-A influenza viruses.
Influenza infection is an acute fever-like virus infection
of the respiratory tract. The influenza virus and its toxin
can lead to a serious inflammation of the bronchial mucosa
and a damage of the tract.
The influenza viruses belong to the family of
Orthomyxoviridae that have linear segmented (8) negative
ssRNA genome with lipid envelope. Total genome length is
12000-15000 nucleotides (nt). The largest segment is
2300-2500 nt; the second largest is 2300-2500 nt; the third
is 2200-2300 nt; the fourth is 1700-1800 nt; the fifth is
1500-1600 nt; the sixth is 1400-1500 nt; the seventh is
1000-1100 nt; the eighth is 800-900 nt. Genome sequence has
terminal repeated sequences; repeated at both ends.
The virion envelope could be spherical, or filamentous with
diameter of 50-120 nm, or 20 nm and 200-300(-3000) nm long.
About 500 spikes are dispersed evenly over all the surface
(i.e. hemagglutininesterase (HEF)), or dispersed equally
over all the surface, but the various types are in clusters
(i.e. hemagglutinin (HA) the major glycoprotein is
interposed irregularly by clusters of neuraminidase (NA),
with ratio of HA to NA about 4-5 to 1).
The Orthomyxoviridae family is divided into three types :
A,B,C.
Type A influenza viruses
are further divided into subtypes according to their
Hemagglutinin (HA) and Neuraminidase (NA) proteins.
Currently 15 (HA) subtypes and
9(NA) subtypes have been identified.
Type B influenza viruses
produce less serious disease than does influenza type A and
are not categorized as by H or N type as Influenza A is.
Type C influenza viruses
were first isolated in 1949 and are not known to be
responsible for epidemics
The infection in human mostly results from a droplet
infection and appears as an epidemic which sometimes can be
of pandemic proportions. After an incubation time of 1 - 3
days the symptoms appear suddenly- fast raise of
temperature, often accompanied by shivering, the leading
symptom of catarrhal inflammation appears, contributing to
the clinical course of painful dry cough, tracheitis,
laryngitis and frequent rhinitis and conjunctivitis.
The appearance new Influenza epidemics and pandemics are
facilitated by an antigen variability mainly in the HA and
NA. In the past century, three major influenza epidemics
have occurred:
1918-1919:
( Spanish Flu, A (H1N1), 20-50 million deaths worldwide,
nearly half were young, healthy adults.
1957-1958:
Asian Flu (A (H2N2),1st identified
in China Feb. 1957, 70,000 deaths in the United States.
1968-1969:
Hong Kong Flu (A (H3N2), 1st detected
in Hong Kong early 1968, virus still circulating today.
The 1997 outbreak of H5N1 avian influenza (or bird flu)
in humans in Hong Kong caused alarm because it involved
highly pathogenic strains of an influenza subtype A to which
humans lack immunity.
The H5N1 infected 18 humans, 6 of whom died (death
rate of about 70 percent). Most of these cases occurred from
contact with infected poultry or contaminated surfaces;
however, it is thought that a few cases of human-to-human
spread of H5N1 have occurred.
Avian influenza is an infectious
disease of birds caused by type A strains of the influenza
virus. The disease, first identified in Italy more than 100
years ago, now occurs worldwide. Infection triggers a wide
spectrum of symptoms in birds, ranging from mild illness to
a highly contagious and rapidly fatal disease resulting in
severe epidemics. In the H5N1 bird flu in Hong Kong in 1997,
patients had developed symptoms of fever, sore throat, cough
and, in several of the fatal cases, severe respiratory
distress secondary to viral pneumonia. Previously healthy
adults and children, and some with chronic medical
conditions, were affected.
More recently,
outbreaks of avian influenza H5N1 occurred among poultry in
eight countries in Asia (Cambodia, China, Indonesia, Japan,
Laos, South Korea, Thailand, and Vietnam) during late 2003
and early 2004. At that time, more than 100 million birds in
the affected countries either died from the disease or were
killed in order to try to control the outbreak. By March
2004, the outbreak was reported to be under control.
Beginning in late June 2004, however, new deadly outbreaks
of influenza H5N1 among poultry were reported by several
countries in Asia (Cambodia, China, Indonesia, Malaysia
[first-time reports], Thailand, and Vietnam ). It is
believed that these outbreaks are ongoing.
Human infections of avian influenza H5N1 however, have been
reported in Cambodia (1case/1death) Thailand (17cases/1
death) and Vietnam (51cases/ 33deaths) during both of these
outbreak periods.
Hemagglutinin (HA) is a surface glycoprotein on Influenza A
responsible for binding to N-AcetylNeuraminic Acid (NeuNAc)
or commonly Sialic acid on host cell surface receptors. The
Influenza viruses form the A virus group have principally
similar morphological, chemical and biological features. The
differentiation of the types is possible by the different
antigenicity of their nucleo- and matrix proteins that have
type-specific antigenicity. However, the essential
immunodominant antigens and primary targets in diagnosis are
the hemagglutinin (HA) and the neuraminidase (NA) antigens.
Screening for type-specific anti-HA or anti-NA antibodies
has also been proved to be useful method in clinical
identification of different influenza strains.
For
the detection of antibodies to H5 strain of Avian Influenza,
this H5-HA(Ab) kit utilizes solid phase, two-step incubation
blocking Enzyme-linked immunosorbent assays (ELISA) in which
polystyrene microwell strips are pre-coated with monoclonal
anti-H5 antibodies (anti-H5”mAb”). The tested serum/plasma
sample is incubated with a fixed amount of H5 antigen and
anti-H5 antibody conjugated to the enzyme horse-radish
peroxidase (HRP-Conjugate), and the amount of antibody is
determined by measuring the amount of unneutralized antigen.
Assay principle scheme: Blocking ELISA
Ab(p)+Ag+Ab(s) → [Ab(p)-Ag-Ab(s)
] +(Ab)ENZ → [Ab(p)-Ag-Ab(s)
] → No color ( + )
Ab(p)+Ag
→ [Ab(p)-Ag ] +(Ab)ENZ →
[Ab(p)-Ag – (Ab)ENZ ] → Blue →Yellow Color ( - )
Incubation1
Immobilized Complex Incubatiuon2 Immobilized
Complex Coloring Results
60 min.
30min.
15 min.
Ab(p)–pre-coated
H5 anti-HA”mAb”;
Ab(s)–H5
anti-HA specific antibodies in sample;
Ag –
H5 virus antigen;
(Ab)ENZ–HRP
conjugated H5 anti-HA”mAb”;
96 Tests
l
MICROWELL PLATE
1plate
Blank microwell strips fixed on white strip holder.
The plate is sealed in aluminum pouch with desiccant.
8×12
or 12×8-well
strips per plate.
Each well
contains monoclonal antibodies (anti-H5”mAb”).
The microwell strips can be broken to be used separately.
Place unused wells in the plastic sealable storage bag
together with the desiccant and return to 2~8℃.
l
NEGATIVE CONTROL
1vial
Yellowish liquid filled in a vial with green screw cap
0.5ml per vial.
Protein-stabilized buffer tested non-reactive for antibodies
to H5 influenza-A viruses.
Preservatives: 0.1% ProClin 300.
Ready to use
as supplied.
Once open, stable for one month at 2-8℃.
l
POSITIVE CONTROL
1vial
Red-colored color liquid filled in a vial with red screw
cap.
0.5ml per vial.
Protein-stabilized buffer tested reactive for antibodies to
HA of H5 influenza-A viruses. Preservatives: 0.1% ProClin
300.
Ready to use as supplied.
Once open, stable for one month at 2-8℃.
l
HRP-CONJUGATE REAGENT
1vial
Red-colored liquid filled in a white dropper vial with red
screw cap.
12ml per vial.
Horseradish peroxidase-conjugated H5 specific anti-H5”mAb”
(monoclonal).
Ready to use as supplied.
Once open, stable for one month at 2-8℃.
l
ANTIGEN REAGENT
1vial
Yellowish liquid filled in white vial with blue screw cap.
6ml per vial.
H5 Antigen diluted in protein-stabilized buffer.
Ready to use as supplied.
Once open, stable for one month at 2-8℃.
l
STOCK WASH BUFFER
1bottle
Colorless liquid filled in a clear bottle with white screw
cap.
50ml per bottle.
PH 7.4 20 × PBS (Containing
Tween-20 as a detergent).
DILUTE BEFORE USE
-The concentration must be
diluted 1 to 20 with distilled/deionized water before
use.
Once diluted, stable for one week at room temperature
or for two weeks at 2-8℃.
l
CHROMOGEN SOLUTION A
1vial
Colorless liquid filled in a white dropper vial with green
screw cap.
6ml per vial.
Urea peroxide solution.
Ready to use as supplied.
Once open, stable for one month at 2-8℃.
l
CHROMOGEN SOLUTION B
1vial
Colorless liquid filled in a black dropper vial with black
screw cap.
6ml per vial.
TMB solution. Tetramethylbenzidine dissolved in citric acid.
Ready to use as supplied.
Once open, stable for one month at 2-8℃.
l
STOP SOLUTION
1vial
Colorless liquid filled in a white dropper vial with yellow
screw cap
6ml per vial.
Diluted sulfuric acid solution (2.0M H2SO4).
l
PLASTIC SEALABLE BAG
1unit
For enclosing the strips not in
use.
l
CARDBOARD PLATE COVER
1sheet
To cover the plates during incubation and prevent
evaporation or contamination of the wells.
l
PACKAGE INSERTS
1copy
1.
Freshly distilled or deionized water.
2.
Disposable gloves and timer.
3.
Appropriate waste containers for potentially contaminated
materials.
4.
Disposable V-shaped troughs.
5.
Dispensing system and/or pipette (single or multichannel),
disposable pipette tips
6.
Absorbent tissue or clean towel.
7.
Dry incubator or water bath, 37±0.5℃.
8.
Microshaker for dissolving and mixing conjugate with
samples.
9.
Microwell plate reader, single wavelength 450nm or dual
wavelength 450nm and 630nm.
10.
Microwell aspiration/wash system.
1.
Sample Collection:
Either fresh serum or plasma
samples can be used for this assay. Blood collected by
venipuncture should be allowed to clot naturally and
completely – the serum/plasma must be separated from the
clot as early as possible as to avoid hemolysis of the RBC.
Care should be taken to ensure that the serum samples are
clear and not contaminated by microorganisms. Any visible
particulate matters in the sample should be removed by
centrifugation at 3000 RPM for at least 20 minutes at room
temperature, or by filtration on 0.22u filters. Plasma
samples collected into EDTA, sodium citrate or heparin may
be tested, but highly lipaemic, icteric, or hemolized
samples should not be used as they could give erroneous
results in the assay. Do not heat inactivate samples. This
can cause sample deterioration.
2.
Transportation and Storage:
Store samples at 2-8℃.
Samples not required for assaying within 3 days should be
stored frozen (-20℃
or lower).Multiple freeze-thaw cycles should be avoided. For
shipment, samples should be packaged and labeled in
accordance with the existing local and international
regulations for transport of clinical samples and
ethological agents.
1
1.
A good washing procedure is essential to obtain correct and
precise analytical data.
2.
It is therefore recommended to use a good quality ELISA
microplate washer, maintained at the best level of washing
performances. In general, no less than 5 automatic washing
cycles with dispensing of 350-400μl/well, are sufficient to
avoid false positive reactions and high background (all
wells turn yellow).
3.
To avoid cross-contaminations of the plate with sample or
HRP-conjugate, after incubation do not discard the content
of the wells, but allow the plate washer to aspirate it
automatically.
4.
Anyway, we recommend calibrating the washing system on the
kit itself in order to match the declared analytical
performances. Assure that the microplate washer’s liquid
dispensing channels are not blocked or contaminated, and
sufficient volume of Wash buffer is dispensed each time into
the wells.
5.
In case of manual washing, we suggest to perform at least
5cycles, dispensing 350-400μl/well and aspirating the liquid
for 5times. If poor results (high background) are observed,
increase the washing cycles or soaking time per well.
1.
In any case, the liquid aspirated out the strips should be
treated with a sodium hypochlorite solution(final
concentration of 2.5%) for 24 hours, before liquids are
disposed in an appropriate way.
2.
The concentrated Washing solution should be diluted 1
to 20 before use. For one plate, mix 50 ml of the
concentrate with 950ml of water for a final volume of 1000ml
diluted Wash Buffer. If less than a whole plate is used,
prepare the proportional volume of solution.
The
components of the kit will remain stable through the
expiration date indicated on the label and package when
stored between 2-8
℃,
do not freeze. To assure maximum performance of this
H5-HA(Ab) ELISA kit, during storage, protect the reagents
from contamination with microorganism or chemicals.
This kit is
intended
FOR IN VITRO
USE ONLY
FOR
PROFESSIONAL USE ONLY
The ELISA
assay is a time and temperature sensitive method. To avoid
incorrect result, strictly follow the test procedure steps
and do not modify them.
1.
Do not exchange reagents from different lots, or use
reagents from other commercially available kits. The
components of the kit are precisely matched as to achieve
optimal performance during testing.
2.
Make sure that all reagents are within the validity
indicated on the kit box and are of the same lot. Never use
reagents beyond the expiry date stated on reagents labels or
on the kit box.
3.
CAUTION - CRITICAL STEP:
Allow the reagents and samples to stabilize at room
temperature (18-30℃)
before use. Shake reagent gently before, and return to 2-8℃
immediately after use.
4.
Use only sufficient volume of sample as indicated in the
procedure steps. Failure to do so, may cause in low
sensitivity of the assay.
5.
Do not touch the bottom exterior of the wells; fingerprints
or scratches may interfere with microwell reading.
6.
When reading the results, ensure that the plate bottom is
dry and there are no air-bubbles inside the wells.
7.
Never allow the microplate wells to dry after the washing
step. Immediately proceed to the next step. Avoid the
formation of air-bubbles when adding the reagents.
8.
Avoid assay steps long time interruptions. Assure same
working conditions for all wells.
9.
Calibrate the pipette frequently to assure the accuracy of
samples/reagents dispensing. Always use different disposal
pipette tips for each specimen and reagents as to avoid
cross-contaminations. Never pipette solutions by mouth.
10.
The use of automatic pipettes is recommended.
11.
Assure that the incubation temperature is 37℃
inside the incubator.
12.
When adding samples, avoid touching the well’s bottom with
the pipette tip.
13.
When reading the results with a plate reader, it is
recommended to determine the absorbance at 450nm or at 450nm
with reference at 630nm.
14.
All specimens from human origin should be considered as
potentially infectious.
15.
Materials from human origin may have been used in the kit.
These materials have been tested with tests kits with
accepted performance and found negative for antibodies to
HIV ½, HCV, TP and HBsAg. However, there is no analytical
method that can assure that infectious agents in the
specimens or reagents are completely absent. Therefore,
handle reagents and specimens with extreme caution as if
capable of transmitting infectious diseases. Strict
adherence to GLP (Good Laboratory Practice) regulations can
ensure the personal safety. Never eat, drink, smoke, or
apply cosmetics in the assay laboratory.
16.
Bovine derived sera may have been used in this kit. Bovine
serum albumin (BSA) and fetal calf sera (FCS) are derived
from animals from BSE/TSE free-geographical areas.
17.
The pipette tips, vials, strips and sample containers should
be collected and autoclaved for 1hour at 121℃
or treated with 10% sodium hypochlorite for 30minutes to
decontaminate before any further steps for disposal.
18.
The Stop solution (2M H2SO4 ) is a
strong acid. Corrosive. Use it with appropriate care. Wipe
up spills immediately or wash with water if come into
contact with the skin or eyes. ProClin 300 used as a
preservative can cause sensation of the skin.
19.
The enzymatic activity of the HRP-conjugate might be
affected from dust, reactive chemical, and substances like
sodium hypochlorite, acids, alkalins etc. Do not perform the
assay in the presence of such substances.
20.
Materials Safety Data Sheet (MSDS) available upon request.
21.
If using fully automated microplate processing system,
during incubation, do not cover the plates with the plate
cover. The tapping out of the remainders inside the plate
after washing, can also be omitted.
Step1
Reagents preparation: Allow
the reagents to reach room temperature (18-30°C). Check the
Wash buffer concentrate for the presence of salt crystals.
If crystals have formed in the solution, resolubilize by
warming at 37℃ until crystals dissolve. Dilute the stock
Wash Buffer 1 to 20 with distilled or deionized water. Use
only clean vessels to dilute the buffer.
Step2
Numbering Wells:
Set the strips needed in strip-holder and
number sufficient number of wells including three Negative
control (e.g. B1, C1, D1),
two Positive control
(e.g. E1, F1) and one Blank (e.g.
A1, neither samples nor Antigen reagent or HRP-Conjugate
should be added into the Blank well). Use only number of
strips required for the test.
Step3
Adding
Sample: Add 50ml
of Positive control, Negative control, Samples into their
respective wells. Note: Use a separate
disposal pipette
tip for each specimen, Negative
Control and Positive Control to avoid cross-contamination.
Step4 Adding
Antigen Reagent: Add 50ml
Antigen Reagent to each well except the Blank and mix by
tapping the plate gently.
Step5 Incubating:
Cover the plate with the plate cover and incubate for 60
minutes at 37℃.It
is recommended to use water tank to assure the temperature
stability and humidity during the incubation. If dry
incubator is used, do not open the door frequently.
Step6 Washing:
At the end of the incubation, remove and discard the plate
cover. Wash each well 5times with diluted Wash
buffer. Each time allow the microwells to soak for 30-60
seconds. After the final washing cycle, turn the strips
plate down onto blotting paper or clean towel, and tap the
plate to remove any remainders.
Step7 Adding HRP-Conjugate:
Add 100ml HRP-Conjugate
to each well except the Blank and mix by tapping the plate
gently.
Step8 Incubating:
Cover the plate with the plate cover and incubate for 30
minutes at 37℃.It
is recommended to use water tank to assure the temperature
stability and humidity during the incubation. If dry
incubator is used, do not open the door frequently.
Step9 Washing:
At the end of the incubation, remove and discard the plate
cover. Wash each well 5times with diluted Wash
buffer as in Step6.
Step10 Coloring:
Dispense 50ml
of Chromogen A and 50ml
Chromogen B solution into each well including the Blank,
and mix by tapping the plate gently. Incubate the plate at
37℃
for 15minutes avoiding light. The enzymatic reaction
between the Chromogen solutions and the HRP-Conjugate
produces blue color in Negative control and Negative sample
wells.
Step11 Stopping
Reaction: Using a multichannel pipette or manually
add 50ml
Stop Solution into each well and mix gently. Intensive
yellow color develops in Negative control and Negative
sample wells.
Step12
Measuring the Absorbance:
Calibrate the plate reader with the Blank well and read the
absorbance at 450nm.If a dual filter instrument is
used, set the reference wavelength at 630nm.
Calculate the Cut-off value and evaluate the results. (Note:
read the absorbance within 10 minutes after
stopping the reaction)
Each microplate should be considered
separately when calculating and interpreting results of the
assay, regardless of the number of plates concurrently
processed. The results are calculated by relating each
sample optical density (OD) value to the Cut-off value
(C.O.) of the plate. If the Cut-off reading is based on
single filter plate reader, the results should be calculated
by subtracting the Blank well OD value from the print report
values of samples and controls. In case the reading is based
on Dual filter plate reader, do not subtract the Blank well
OD from the print report values of samples and controls.
1.
Calculation of Cut-off value:
Cut-off value (C.O.) = *Nc x 0.5
*Nc = the mean absorbance value for three negative
controls.
Important: If the mean OD value of the negative control is
higher than 2.500, take it as 2.500 .If lower than 2.500,
see the Quality control range.
If one of the Negative control values does not meet
the Quality control range specifications, it should be
discarded and the mean value is calculated again using the
remaining two values. If more than one control OD value does
not meet the Quality control range specifications, the test
is invalid and must be repeated.
2. Quality control range:
The
test results are valid if the Quality Control criteria are
verified. It is recommended that each laboratory must
establish appropriate quality control
system with quality control material similar to or identical
with the patient sample being analyzed
1.
The absorbance of the Blank
well, which contains only Chromogens and Stop solution, is
less than 0.080 at 450 nm.
2.
The absorbance value OD of the
Positive control must be lower than 0.100 at 450/630nm or at
450nm after blanking.
3.
The absorbance value OD of the
Negative control must equal to or higher than 1.000 at
450/630nm or at 450nm after blanking.
3. Interpretations of the results:
Negative Results (S/C.O.
≥1):
samples giving an absorbance higher than or equal to the
Cut-off value are considered negative, which indicates that
antibodies to the hemagglutinin of H5 avian influenza have
not been detected with this kit.
Positive Results (S/C.O. < 1):
samples giving an absorbance less than the Cut-off value are
considered initially reactive, which indicates that
antibodies to hemagglutinin of H5 avian influenza have
probably been detected with this kit. Any initially reactive
samples must be retested in duplicates. Repeatedly reactive
samples can be considered positive for H5 HA specific
antibodies.
Borderline:
Samples with absorbance to Cut-off ratio
between 0.9 and 1.00 are considered borderline samples and
retesting is recommended. Repeatedly positive samples can be
considered positive for H5 HA antibodies.
1.
Non-repeatable positive result
may occur due to the general biological and biochemical
characteristics of ELISA assays. The test is designed to
achieve performance characteristics of high sensitivity and
specificity. HA may be undetectable during the early stages
of the disease and in some immunosuppressed individuals.
2.
Any positive results must be
interpreted in conjunction with patient clinical
information and other laboratory testing results.
3.
Common sources for mistakes:
kits beyond the expiry date, bad washing procedures,
contaminated reagents, incorrect assay procedure steps,
insufficient aspiration during washing, failure to add
samples or reagents, equipment, timing, volumes, sample
nature and quality.
4.
If, after retesting of the
initially reactive samples, the assay results are negative ,
these samples should be considered as non-repeatable (false
positive) and interpreted as negative. As with many very
sensitive ELISA assays, false positive results can occur due
to the several reasons, most of which are related but not
limited to inadequate washing step.
5.
The prevalence of the marker
will affect the assay’s predictive values.
6.
This kit is intended ONLY for
testing of individual serum or plasma samples. Do not use it
for testing of cadaver samples, saliva, urine or other body
fluids, or pooled (mixed) blood.
Please do not use this kit beyond the expiry date indicated
on the kit box and reagent labels.
1.
Values of the Positive or Negative controls ,which are out
of the indicated Quality control range, are indicator of
possible deterioration of the reagents and/or operator or
equipment errors. In such case, the results should be
considered as invalid and the samples must be retested. In
case of constant erroneous results classified as due to
deterioration or instability of the reagents, immediately
substitute the reagents with new ones.
2.
If after mixing of the Chromogen A and B solutions into the
wells, the, the color of the mixture turns blue within few
minutes, do not continue carrying out the testing and
replace the reagents with fresh ones.
REFERENCES:
1.
Fouchier RAM, Schneeberger PM, Rozendaal FW, et al. Avian
influenza A virus (H7N7) associated with human
conjunctivitis and a fatal case of acute respiratory
distress syndrome. Proc Natl Acad Sci 2004. Published online
before print January 26, 2004
2.
Fouchier RAM, Munster V, Wallensten A, et al.
Characterization of a novel influenza A virus hemagglutinin
subtype (H16) obtained from black-headed gulls J Virol 2005
Mar;79(5):2814-22
3.
Horimoto T, Kawaoka Y. Pandemic threat posed by avian
influenza A viruses. Clin Microbiol Rev 2001;14(1):129-49
4.
Keawcharoen J, Oraveerakul K, Kuiken T, et al. Avian
influenza H5N1 in tigers and leopards. Emerg Infect Dis 2004
Dec;10(12)
5.
Luschow D, Werner O, Mettenleiter TC, et al. Protections of
chickens from lethal avian influenza A virus infections by
live-virus hemagglutinin (H5) gene. Vaccine 2001 Jul
20;19(30):4249-59
6.
Monto AS. The threat of an avian influenza pandemic.
(Perspective) N Engl J Med 2005 Jan 27;352(4):323-4
7.
OIE. Highly pathogenic avian influenza. Technical disease
card database. Apr 22, 2002
8.
OIE. Highly pathogenic avian influenza. International Health
Code. Chap 2.7.12. 2004
9.
OIE. Update on avian influenza in animals in Asia (type H5).
Updated frequently
10.
Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, et
al. Characterization of an avian influenza A (H5N1) virus
isolated from a child with a fatal respiratory illness.
Science 1998;279:393–6.
11.
Yuen KY, Chan PKS, Peiris M, Tsang DNC, Que TL, Shortridge
KF, et al. Clinical features and rapid viral diagnosis of
human disease associated with avian influenza A H5N1 virus.
Lancet 1998;351:467–71.
|
SUMMARY OF THE MAJOR COMPONENTS OF THE KIT: |
|
Microwell plate |
One/ 96 wells |
|
Negative/ Positive control |
One each/ 0.5ml |
|
HRP-Conjugate |
One/ 12ml |
|
Antigen Reagent |
One/ 6ml |
|
Wash Buffer |
One/ 50ml |
|
Chromogen A/B/ Stop Solution |
One each/ 6ml |
|
Note: the components of individual kits are not
interchangeable |
|
SUMMARY OF THE ASSAY PROCEDURE: |
Add Sample
|
50ml |
Add Antigen
Reagent
|
50ml |
|
Incubate 37℃ |
60minutes |
|
Wash |
5times |
|
Add HRP-Conjugate |
50ml |
|
Incubate 37℃ |
30minutes |
|
Wash |
5times |
|
Coloring |
50ml
A +
50ml B |
|
Incubate 37℃ |
15minutes |
|
Stop the reaction |
50ml
stop solution |
|
Read the absorbance |
450nm or 450/630nm |
