INFECTIOUS BRONCHITIS VIRUS ANTIBODY TEST KIT

INFECTIOUS BRONCHITIS VIRUS ANTIBODY TEST KIT

Translation Procedure:
QC5345
Version 1.0
07/03/2002
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Assay Procedure:
Warm reagents to room
temperature. Mix all reagents by
inverting and swirling.
Procedimiento de ensayo:
Caliente los reactivos a
temperatura ambiente.
A continuación, invierta y agite el
tubo para mezclarlos.
Description du test :
Ramenez les réactifs à la
température ambiante. Mélangez
tous les réactifs en agitant bien.
Testanweisung:
Erwärmen Sie die Reagenzien auf
Zimmer-temperatur. Mischen Sie
alle Reagenzien durch Invertieren
und Wirbeln.
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Dilute 4X Sample Diluent by
mixing 1 part with 3 parts
deionized water. Mix well. For
example, 30ml 4X Sample
Diluent plus 90ml deionized water.
Dilute 20X Wash Solution by mixing
1 part with 19 parts deionized water.
Mix Well. For example, 25ml of 20X
Wash Solution plus 475ml deionized
water.
Shake to suspend Negative,
Positive and test samples.
Dilute Samples in a ratio of 2 µl
sample with each 800 µl Sample
Diluent. Mix well by pipetting 4
times with 100µl displacement
Dispense 100µl of Negative to
duplicate wells. Dispense 100µl of
Positive to duplicate wells. Dispense
100µl diluted test sample per well. Let
stand 30 minutes at room temperature.
Mezcle bien una parte de
diluyente de muestra 4X con tres
partes de agua desionizada. Por
ejemplo, utilice 30 ml de diluyente
de muestra 4X y 90 ml de agua
desionizada.
Mezcle bien una parte de Solución
Lavada 20X con 19 partes de agua
desionizada. Por ejemplo, utilice 25
ml de Solución Lavada 20X y 475 ml
de agua desionizada.
Agite para suspender muestras
negativas, positivas y de
prueba.
Diluya Muestras en una proporción de
2 µl de muestra por cada 800 µl de
diluyente de muestra. Mezcle bien
pipeteando cuatro veces con un
desplazamiento de 100 µl.
Diluez 20 fois la solution de nettoyage
en mélangeant 1 volume de solution
de nettoyage avec 19 volumes d’eau
désionisée. Mélangez bien. A titre
d’exemple, mélangez 25 ml de
solution de nettoyage avec 475 ml
d’eau désionisée.
Agitez pour suspendre les
échantillons négatifs, positifs
et témoins.
Diluez les échantillons témoins dans
un rapport de 2 µl d’échantillon par
800 µl de diluant d’échantillon.
Mélangez bien en pipetant 4 fois par
groupe de 100 µl.
Vierta, en dos pocillos, 100 µl de muestra
negativa. Vierta, en dos pocillos, 100 µl
de muestra positiva. Vierta 100 µl de
muestra
de prueba diluida en cada pocillo. Deje
reposar 30 minutos a temperatura
ambiente.
Verdünnen Sie 20X Wash Solution
durch Mischen eines Teils mit
neunzehn Teilen Deionat. Gut
mischen. Beispielgröße: 25 ml 20X
Wash Solution mit 475 ml Deionat.
Schütteln Sie, um Negativ,
Positiv und die Messproben.
Verdünnen die Messproben im
Verhältnis 2 µl pro Probe für jeweils
800 µl Sample Diluent. Gut mischen.
Führen Sie vier Pipettierungen durch,
jeweils um 100 µl versetzt.
Diluez 4 fois le diluant d’échantillon
en mélangeant 1 volume de diluant
avec 3 volumes d’eau désionisée.
Mélangez bien. A titre d’exemple,
mélangez 30 ml de diluant
d’échantillon avec 90 ml d’eau
désionisée.
Verdünnen Sie 4X Sample Diluent
durch Mischen eines Teils mit drei
Teilen Deionat. Gut mischen.
Beispielgröße: 30 ml 4X Sample
Diluent mit 90 ml Deionat.
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Empty plate. Wash by dispensing 300ul Wash
Solution per well. Empty plate and tap out excess
on paper towel. Repeat two more times. Do not
allow plate to dry between washing steps.
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Immediately dispense 100µl
Conjugate per well.
Let stand 30 minutes at room
temperature.
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Placez 100 µl de solution négative pour
dédoubler les puits. Placez 100 µl de
solution positive pour dédoubler les puits.
Placez 100 µl d’échantillon témoin par
puits. Laissez reposer 30 minutes à
température ambiante.
Geben Sie jeweils 100 µl Negativs an
gleichförmige Schächte ab. Geben Sie
jeweils 100 µl Positivs an gleichförmige
Schächte ab. Geben Sie ebenso 100 µl
verdünnte Messprobe pro Schacht ab. 30
Minuten bei Zimmertemperatur lagern.
Immediately dispense 100µl
Substrate per well. Let stand 30
minutes at room temperature.
Do not empty plate.
Immediately dispense 100µl
Stop Solution per well.
Read plate using dual wavelength microplate
reader blanked on air. Use a 405 nm or 410
nm as primary filter. Use a 630 nm or 650
nm as reference filter.
No vacíe la placa. Vierta
inmediatamente 100 µl de
Solucción de Cesacion en
cada pocillo.
Lea la placa utilizando un lector de microplacas
con longitud de onda dual (emplear aire como
blanco). Use un filtro primario de 405 ó 410
nm. Use un filtro de referencia de 630 ó 650
nm.
Ne videz pas la plaque.
Placez immédiatement
100 µl de solution d’arrêt
par puits.
Analysez la plaque en utilisant un lecteur de
microplaque à deux longueurs d’ondes à
blocage d’air. Utilisez un filtre principal de
405 nm ou 410 nm. Utilisez la valeur 630 nm
ou 650 nm comme filtre de référence.
Vacíe la placa. Lave cada pocillo con 300 µl de
Solución Lavada. Vacíe la placa y sacúdala sobre
una toallita de papel para eliminar el líquido
sobrante. Repita este procedimiento dos veces más.
No permita que la placa se seque entre lavados.
Vierta inmediatamente 100 µl de
conjugado en cada pocillo. Deje
reposar 30 minutos a temperatura
ambiente.
Lave la placa como se
indica en el paso 7.
Videz la plaque. Lavez-la en plaçant 300 µl de
solution de nettoyage par puits. Videz la plaque et
faites tomber tout résidu sur de l’essuie tout en
papier. Répétez l’opération deux fois. Ne laissez pas
la plaque sécher entre deux lavages.
Placez immédiatement
100 µl de conjugué. Laissez
reposer 30 minutes à
température ambiante.
Lavez la plaque comme à
l’étape 7.
Placez immédiatement
100 µl de substrat par puits. Laissez
reposer 30 minutes à température
ambiante.
Leeren Sie den Boden. Waschen Sie durch Ausgabe
von 300 µl Wash Solution pro Schacht nach. Leeren
Sie den Boden und entnehmen Sie den Überschuss
mit einem Papierhandtuch. Wiederholen Sie den
Vorgang zweimal. Lassen Sie den Boden nicht
zwischen den Waschvorgängen trocknen.
Geben Sie sofort danach 100 µl
Conjugate pro Schacht ab. 30
Minuten bei Zimmertemperatur
lagern.
Waschen Sie den Boden,
wie in Schritt 7 beschrieben.
Geben Sie sofort danach 100 µl
Leeren Sie den Boden nicht.
Substrate pro Schacht ab. 30 Minuten Geben Sie sofort 100 µl Stop
bei Zimmertemperatur lagern.
Solution pro Schacht ab.
for use in CHICKEN
12
Wash plate as in step 7.
Vierta inmediatamente 100 µl de
sustrato en cada pocillo. Deje
reposar 30 minutos a temperatura
ambiente.
INFECTIOUS
BRONCHITIS
VIRUS
ANTIBODY
TEST KIT
Lesen Sie den Boden mit einem Luft
leergesetzten Mikroplattenlesers mit zwei
Wellenlängen ab. Verwenden Sie
Primärfilter von 405 nm oder 410 nm und
Referenzfilter von 630 nm oder 650 nm.
:
IBV-1000, IBV-0500, IBV-0200
PRODUCT CODE
AFFINITECH, LTD.
2308 SE 28th St., Suite 2, Bentonville, AR 72712 (479)464-0991 FAX (479)464-0993
U.S. VET LIC. No. 450
REAGENTS SUPPLIED
NAME AND INTENDED USE
For the detection of Infectious Bronchitis Virus (IBV) antibodies in chicken serum.
PRINCIPAL OF ASSAY
Infectious Bronchitis Virus (IBV) is a clinically acute, highly contagious viral disease
affecting chickens of all ages. IBV has an incubation period of only 24 to 72 hours. The
disease affects the respiratory and urogenital tract of chickens. Young chickens exhibit
acute respiratory disease signs and lesions in the trachea. Mortality may be as high as
100%1. Antibodies to avian viruses in serum can be detected by the Enzyme Linked
Immunosorbant Assay (ELISA) 2. The levels of IBV antibodies in serum, as well as the
immune status of entire flocks can be monitored using ELISA.3
Inactivated antigen is bound to a microwell. Diluted sera is added to the microwell and
incubated. Any anti-IBV antibodies present in the serum will bind to the IBV on the
microwell forming an IBV antigen-antibody complex. After washing away any unbound
material, anti-Chicken IgG Alkaline Phosphatase conjugate is added to the microwell.
The conjugate will bind to the IBV antigen-antibody complex. After incubation any
unbound conjugate is removed by washing. Enzyme substrate is then added to the
microwell. The substrate reacts with any Alkaline Phosphatase present and forms a
yellow product. In the final step the reaction is stopped, fixing the color. The intensity
of the color is measured photometrically at 405 - 410 nm.
Antigen Wells
12 x 8 strips, Wells coated with IBV
Sample Diluent (4X)
Red buffer with protein stabilizers,
Prepare 1X by adding 30ml of 4X Sample
Diluent plus 90ml deionized water.
Wash Solution (20X)
Opaque solution, Prepare 1X by adding
25ml of 20X Wash Solution plus
475ml deionized water.
Positive - Supplied Ready to Use.
Value set at 100 EU, ++ label on cap.
Negative - Supplied Ready to Use.
Negative, - label on cap
Conjugate
Green solution, α-chicken IgG Alkaline
Phosphatase
Substrate
Clear solution, p-Nitrophenyl phosphate
Stop Solution
Clear solution, 3.0 M NaOH
1. Handle all biological reagents as if they may transmit disease.
2. Although the virus has been deactivated, handle as if they may transmit disease. It should be
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IBV-1000
10 plates
(960 wells)
IBV-0500
5 plates
(480 wells)
IBV-0200
2 plates
(192 wells)
1.
2.
1 X 320 ml
1 X 160 ml
1 X 60 ml
3.
1 X 250 ml
1 X 125 ml
1 X 50 ml
1 X 12 ml
1 X 6 ml
1 X 2.5 ml
1 X 12 ml
1 X 6 ml
1 X 2.5 ml
1 X 120 ml
1 X 60 ml
1 X 25 ml
1 X 120 ml
1 X 60 ml
1 X 25 ml
1 X 120 ml
1 X 60 ml
1 X 25 ml
4.
Antigen Coated Wells
1 plate of 12 × 8 strips
Sample Diluent (1X)
120 ml
Wash Solution (1X)
500 ml
Positive
100 µl (do NOT dilute)
Negative
100 µl (do NOT dilute)
Conjugate
12 ml
Substrate
12 ml
Stop Solution
12 ml
Positive and Negative controls must be included with each test run.
The average absorbance value of the Positive Control wells minus the average
Negative Control wells must be 0.7 O.D. or greater.
The average absorbance value of the Negative Control wells must be less than 0.4
O.D. Follow manufacturer’s instructions on blanking the instrument.
If any of the above criteria are not met, the results are invalid and the test should be
repeated.
CALCULATION OF RESULTS
Subtract the average absorbance of the Negative Control wells from the average
absorbance value of the Positive Control and samples. Note: absorbance values of the
controls or samples ran in duplicate should be averaged. The reactivity of each sample
can be calculated as shown here.
Ave. Abs. Test Sample – Ave. Abs. Negative
= Sample to Positive Ratio (Sp)
Ave. Abs. Positive – Ave. Abs. Negative
REAGENT AMOUNTS REQUIRED FOR EACH PLATE
PRECAUTIONS AND PROCEDURAL NOTES
assumed that the sera used in the experiment might contain active virus and should be handled
and disposed as if they are infectious.
Buffers and solutions used or waste generated during the test should be decontaminated with
bleach before disposal.
Do not allow reagents to contact the skin or eyes. If contact occurs, wash with copious amounts
of water.
Certain test reagents contain Proclin 300 as a preservative. When disposing of solutions
containing Proclin 300, flush drains with large volumes of water.
Do not pipet by mouth.
Store reagents at 2 to 7°C. Remove quantity of reagents needed and bring to room temperature 20
to 30 minutes before use and return to 2 to 7°C promptly after use.
Positive and Negative are included in the kit READY TO USE. DO NOT DILUTE.
IBV coated microwell strips should be stored with the desiccant in the resealable pouch provided
and returned to 2 to 7°C immediately after the required number are removed for testing.
Take care to prevent the contamination of kit components.
Do not mix or interchange reagents from different lots or use reagents beyond the expiration date.
Incubation temperature above or below the recommended 20 to 30°C and incubation times other
than those indicated may give erroneous results.
The ELISA assay is a very sensitive technique. Pipetting, incubation and temperature errors will
be magnified. Cross contamination between reagents or between microwells will invalidate the
test.
The washing procedure is very important and requires special attention. An improperly washed
microwell may cause erroneous results.
QUALITY CONTROL
Sp x (100) = ELISA Unit (EU)
ELISA UNITS INTERPRETATION
IBV ELISA VALUES
Less than 10 EUs
INTERPRETATION
Negative for antibodies to IBV
Greater than 10 EUs
Positive for antibodies to IBV
REFERENCES
MATERIALS REQUIRED
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8.
Precision pipets for dispensing 2, 100, and 800 µl
Multichannel pipet for dispensing up to 100 µl
Timer
Graduated cylinders
Distilled water
Plate washing apparatus
Dilution tubes
Strip or plate reader capable of reading dual absorbance at 405 - 410 nm with a 630-650 nm
reference filter and a range value up to 3.0 O.D.
SPECIMEN COLLECTION
Whole blood (at least 0.5 ml) should be collected by accepted techniques. The serum is separated from the
clot and refrigerated, 2 to 7°C, for short-term storage or stored frozen for long-term storage. Avoid multiple
freeze thaw cycles. Specimens containing visible particulate matter should be clarified by centrifugation
before testing. Grossly contaminated specimens should not be used. (I.e. highly turbid, bacterial
contamination, etc.)
Positive Control value is set at 100 EU.
1.
2.
3.
Purchase H.G., Arp L.H., Domermuth C.H., and Pearson, J.E. (eds) A Laboratory Manual for
the Isolation and Identification of Avian Pathogens. Kendall/Hunt, Dubuque, IA.
Snyder D.B., Marquardt W.W., Mallinson E.T., Savae P.K., and Allen D.C. (1984) Rapid
Serological Profiling by Enzyme-Linked Immunosorbent Assay, III. Simultaneous Measurements
of Antibody Titers to Infectious Bronchitis, Infectious Bursal Disease, and Newcastle Disease
Viruses in a Single Serum Dilution. Avian Dis. 28:12-24.
Miers, L.A., R.A. Bankowski, and Y.C. Zee. (1987) Optimizing the enzyme-linked
immunosorbent assay for evaluating immunity of chickens to Newcastle disease. Avian Dis.
27:1112-1125.
Version 1.3
Version Date 05/20/2009