PrioCHECK® BHV-1 gB
ELISA for in vitro detection of antibodies directed against Bovine Herpes Virus in serum and
milk of cattle
50 plate kit for 4400 samples
©Prionics AG
Package Insert
Version 2.0
Introduction
Bovine Herpesvirus 1 (BHV-1), a member of the
subfamily Alphaherpesviridae, can cause a wide
variety of symptoms, including Infectious Bovine
Rhinotracheitis (IBR), Infectious Pustular Vulvovaginitus (IPV) and Infectious Pustular Balanoposthitis (IPB).
Following infection, latency of the virus is established.
As a result of stressful conditions, the latent virus may
be reactivated and shed at irregular intervals.
Because detection of latent BHV-1-carriers is of
importance in control programs and international trade
activities, tests to detect specific antibodies in serum
must be highly sensitive. For that purpose a simple,
specific and highly sensitive gB ELISA has been
developed (PrioCHECK® BHV-1 gB).
The PrioCHECK® BHV-1 gB detects antibodies directed against the surface glycoprotein B of BHV1. By
testing undiluted bovine serum samples, the PrioCHECK® BHV-1 gB ELISA is superior to the 24 hours
neutralization test in detecting low antibody levels. By
testing bulk milk samples, the PrioCHECK® BHV-1 gB
is a simple and convenient tool to determine the
infection status of a herd.
Test Principle
The PrioCHECK® BHV-1 gB is a blocking ELISA. Test
Plates have been coated with BHV-1 antigen. Undiluted or diluted serum samples or undiluted defatted
(bulk) milk samples are incubated overnight in the
wells of a BHV-1-antigen-coated ELISA plate. During
this incubation period, BHV-1-specific antibodies in the
test sample, if present, will react specifically with the
BHV-1 antigen. After this incubation, plates are
washed and incubated with peroxidase-conjugated
Mab (anti-BHV-1 gB). Next, unbound material is
washed away and ready-to-use Chromogen (TMB)
Substrate is added to the wells.
Finally, color development is stopped and measured at
a wavelength of 450 nm.
Kit Components
Store kit at 5±3°C until expiry date. See kit label for
actual expiry date. The shelf life of diluted, opened or
reconstituted components is noted below, when
appropriate. Chemical hazard data are available in
section “Safety Regulations and R&S Statements”
(Appendix II).
Component 1
Test Plate
50 Test Plates.
Component 2
Conjugate (100x)
(100x concentrated, dilute before use)
One vial contains 5.1 ml Conjugate.
Diluted conjugate is not stable, prepare just before
use.
Component 3
Dilution buffer (2x)
(2x concentrated, dilute before use)
One vial contains 500 ml Dilution Buffer.
Shelf life of dilution buffer working solution: 4 hours at
22±3°C.
Component 4
Washing Fluid (200x)
(200x concentrated, dilute before use)
Three vials, each contains 60 ml Washing Fluid.
Shelf life of washing solution: 1 week at 22±3°C.
Component 5
Horse Serum (Ready-to-use)
One vial contains 100 ml Horse Serum.
Component 6
Negative Control (Ready-to-use)
One vial contains 9.0 ml Negative Control.
For in-vitro veterinary diagnostic use only
Store at 5±3°C
Product No.: 7588995
ƒ Pipette tips have to be changed for every pipetting
step.
ƒ Separate solution reservoirs must be used for each
reagent.
ƒ Kit components must not be used after their expiry
date or if changes in their appearance are observed.
ƒ Kit components of different kit lot numbers must not
be used together.
ƒ Demineralized or water of equal quality must be
used for the test.
Component 7
Weak Positive Control (Ready-to-use)
One vial contains 9.0 ml Weak Positive Control.
Component 8
Positive Control (Ready-to-use)
One vial contains 9.0 ml Positive Control.
Component 9
Chromogen (TMB) Substrate (Ready-to-use)
One vial contains 500 ml Chromogen (TMB) Substrate.
Component 10
Stop Solution (Ready-to-use)
One vial contains 500 ml Stop Solution.
Additional Kit Contents:
- Package Insert
- Plate sealers
- Certificate of analysis
Additional Material Required
General:
Laboratory equipment according to national safety
regulations.
Incubation:
Microplate Incubator (reaching at least 50°C).
Analysis of Results:
Plate Reader e.g. Multiscan EX or equivalent.
The reader has to have an appropriate filter set to read
the plates at 450 nm.
Optional:
Plate washer e.g. Tecan EIA Tray Washer or equivalent.
SOLUTIONS TO BE MADE IN ADVANCE
Dilution buffer working solution
The Dilution Buffer (2x) (Component 3) must be diluted
1/2 in demineralized water. Prepare 24 ml per plate
(12 ml Dilution Buffer (2x) and 12 ml demineralized
water). Shelf life of dilution buffer working solution: 4
hours at 22±3°C.
ELISA buffer
Add Horse Serum (Component 5) to dilution buffer
working solution until a final concentration of 10%
(v/v). Prepare 24 ml per plate (2.4 ml Horse Serum
and 21.6 ml dilution buffer working solution). Unused
ELISA buffer can be stored at 5±3°C for up to 24
hours.
Conjugate dilution
Dilute the Conjugate (100x) (Component 2) 1/100 with
ELISA buffer. Prepare 10 ml for one Plate.
Note: The conjugate dilution must be prepared just
before use.
Washing solution
The Washing Fluid (200x) (Component 4) must be
diluted 1/200 in demineralized water and is sufficient
for a final volume of 12 liters of washing solution.
Stability of washing solution: 1 week at 22±3°C.
Note: Commercial available ELISA washers can be
used. If not available, washing of the plates can be
done by dispensing at least 200 - 300 µl of washing
solution to all wells of the plate. Subsequently, empty
the plate and repeat as many times as prescribed. It is
not necessary to soak the plate between washings.
Tap the plate firmly after the last washing.
Note: see Appendix IV for sample preparation
procedure and storage.
Test Procedure
Precautions
National guidelines for working with animal samples
must be strictly followed. The PrioCHECK® BHV-1 gB
must be performed in laboratories suited for this
purpose.
Samples should be considered as potentially infectious
and all items which contact the samples as potentially
contaminated.
Chemical hazard data are available in section “Safety
Regulations and R&S Statements” (Appendix II).
Notes
To achieve optimal results with the PrioCHECK® BHV1 gB, the following aspects must be considered:
ƒ The Test Procedure protocol must be strictly
followed.
ƒ All reagents of the kit must be equilibrated to room
temperature (22±3°C) before use.
DAY 1
INCUBATION OF TEST SAMPLES IN ANTIGEN
COATED WELLS
1.1
1.2
1.3
1.4
1.5
1.6
Dispense 100 µl of ELISA buffer to well A1 and
B1 of the Test Plate (Component 1).
Dispense 100 µl of Negative Control (Component 6) to well C1 and D1.
Dispense 100 µl of Weak Positive Control
(Component 7) to wells E1 and F1.
Dispense 100 µl of Positive Control (Component
8) to wells G1 and H1.
Dispense 100 µl of test samples to the remaining
wells.
- Serum samples can be tested undiluted or titrated by dispensing 2-fold serial dilutions made
in ELISA buffer into the wells.
- Defatted milk samples must be tested undiluted.
Seal the Test Plate.
1
PrioCHECK® BHV-1 gB
1.7
Incubate overnight (16 -18 hours) at 37±1°C.
Note: In case serum test samples have been titrated,
the antibody titre of a sample is defined as the reciprocal of the highest dilution, which gives a PI >40%.
DAY 2
INCUBATION WITH CONJUGATE AND CHROMOGEN (TMB) SUBSTRATE
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
Empty the Test Plate after the incubation period
and wash the plate 6 times with washing solution. Tap the plate firmly after the last washing.
Dispense 100 µl of the diluted conjugate to all
wells.
Seal the Test Plate using the enclosed plate
sealers.
Incubate 60±5 minutes at 37±1°C.
Empty the Test Plate after the incubation period
and wash the plate 6 times with washing solution. Tap the plate firmly after the last washing.
Dispense 100 µl of Chromogen (TMB) Substrate
(Component 9) to all wells.
Incubate 15 minutes at 22±3°C.
Add 100 µl of the Stop Solution (Component 10)
to all wells.
Mix the content of the wells of the Test Plate
prior to measuring.
Note: Start the addition of Stop Solution 15 minutes
after the first well was filled with Chromogen (TMB)
Substrate. Add the Stop Solution in the same order
and at the same pace as the Chromogen (TMB)
Substrate was dispensed.
READING OF THE TEST AND CALCULATING
THE RESULTS
3.1
3.2
3.3
Measure the optical density (OD) of the wells at
450 nm preferable within 15 minutes after colour
development has been stopped.
Calculate the mean OD450 value of wells A1 and
B1 (=OD450 max).
The percentage inhibition (PI) of the controls and
the test sera are calculated according to the formula below.
Note: The OD450 values of all samples are expressed
as Percentage Inhibition (PI) relative to the
OD450 max.
OD450 test sample
PI = 100 – -----------------------------OD450 max
x 100
Validation criteria
4.2
4.3
4.4
Prionics AG shall not be liable for consequential,
incidental, special or any other indirect damages
resulting from economic loss or property damage
sustained by any customer from the use of its products.
Prionics AG and Prionics Lelystad B.V. are ISO
9001:2000 certified companies.
Appendix II
Safety Regulations and R&S Statements
RESULT INTERPRETATION
4.1
4.2
Appendix I
Notice
This manual is believed to be complete and accurate
at the time of publication. In no event shall Prionics AG
be liable for incidental or consequential damage in
connection with or arising from the use of this manual.
Liability
Prionics AG warrants its products will meet their
applicable published specification when used in
accordance with their applicable instructions and
within the declared products life time. Prionics AG
makes no other warranty, expressed or implied. There
is no warranty of merchantability or fitness for a particular purpose. The warranty provided herein and the
data, specifications and descriptions of Prionics AG
products appearing in Prionics AG published catalogues and product literature may not be altered
except by express written agreement signed by an
officer of Prionics AG. Representation, oral or written,
which are inconsistent with this warranty or such
publications are not authorized and if given, should not
be relied upon.
In the event of a breach of the foregoing warranty,
Prionics AG’s sole obligation shall be to repair or
replace, at its option, the applicable product or part
thereof, provided the customer notifies Prionics AG
promptly of any such breach. If after exercising reasonable efforts, Prionics AG is unable to repair or
replace the product or part, then Prionics AG shall
refund to the customer all monies paid for such applicable product or part.
The mean OD450 max must be >1.000.
The mean percentage inhibition of the Negative
Control must be <40%.
The mean percentage inhibition of the Weak
Positive Control must be >40%.
The mean percentage inhibition of the Positive
Control must be >70%.
Not meeting any of these criteria is reason to
discard the results of that specific plate.
Note: If the OD450 of a test sample is higher than the
OD450 max the Percentage Inhibition can be interpreted as 0%.
If the OD450 max is below 1.000 possibly the Chromogen (TMB) Substrate is too cold. In that case warm
the solution to 22±3°C or incubate up to 30 minutes.
If the OD450 max is above 2.000 a shorter incubation
period with the Chromogen (TMB) Substrate is recommended.
Interpretation of the percent inhibition
PI = <40%: Negative for BHV-1 specific antibodies.
PI = ≥40%: Positive for BHV-1 specific antibodies.
National Safety Regulations must be strictly followed.
R&S Statements
Component 1
Test Plate
Hazard Code: This product is not classified according
to EU regulations.
Component 2
Conjugate (100x)
Hazard Code: This product is not classified according
to EU regulations.
Component 3
Dilution Buffer (2x)
Hazard Code: This product is not classified according
to EU regulations.
Component 4
Washing Fluid (200x)
Hazard Code: This product is not classified according
to EU regulations.
Component 5
Horse Serum (Ready-to-use)
Hazard Code: This product is not classified according
to EU regulations.
Component 6
Negative Control (Ready-to-use)
Hazard Code: This product is not classified according
to EU regulations.
Component 7
Weak Positive Control (Ready-to-use)
Hazard Code: This product is not classified according
to EU regulations.
Component 8
Positive Control (Ready-to-use)
Hazard Code: This product is not classified according
to EU regulations.
Component 9
Chromogen (TMB) Substrate (Ready-to-use)
Hazard Code: This product is not classified according
to EU regulations.
Component 10
Stop Solution (Ready-to-use)
Hazard Code: R35: Causes severe burns.
S26: In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice.
S36/37/39: Wear suitable protective clothing, gloves
and eye/face protection.
S45: In case of accident or if you feel unwell, seek
medical advice immediately (show the label on vial).
Appendix III
References
1)
2)
3)
Ackermann, M., S. Belak, V. Bitsch, S. Edwards,
A. Moussa, G. Rockborn, and E. Thiry. 1990.
Vet. Microbiol. 23: 361-363.
J.A. Kramps, J. Magdalena, J. Quak, K. Weerdmeester, M.J. Kaashoek, M.A. Maris-Veldhuis,
F.A.M. Rijsewijk, G. Keil and J.T.van Oirschot.
1994. J. of Clin. Microbiol. 1994. p. 2175-2181.
J.A. Kramps, B. Perrin, S. Edwards and J.T. van
Oirschot. 1996. Vet. Microbiol. 53: 153-161.
Appendix IV
Sample preparation procedure and storage
Serum
- Serum samples can be stored at –20°C before
testing.
- When titrating serum samples, two-fold serial
dilutions should be prepared in ELISA buffer.
Milk
- Defatted milk samples, whether or not containing a
preservative like azide, should be prepared by
centrifugation for 15 minutes at 1000xg. Instead of
centrifugation, samples can be kept refrigerated
for at least 16 hours. The defatted sample can be
taken carefully from underneath the creamy layer
by using a Pasteur pipette.
- Before testing, milk samples can be stored at
–20°C when carefully defatted. Alternatively, milk
samples can be stored at 5±3°C for several days
when a preservative (e.g. azide) has been added.
Contact
Prionics Lelystad B.V.
Platinastraat 33
8211 AR Lelystad
The Netherlands
Tel. +31 320 714000
Fax +31 320 214379
Prionics AG
Wagistrasse 27a
CH-8952 Schlieren-Zurich
Switzerland
Tel. +41 44 200 2000
Fax +41 44 200 2010
www.prionics.com
[email protected]
For our distribution network, please refer to
www.prionics.com.