INSTRUCTIONS FOR USE
For Veterinary Use Only. For In Vitro Use Only.
PrioCHECK™ Bovine PI3 Ab Serum Kit
Immunoenzymatic test for specific detection of anti-PI3 antibodies in bovine serum
Catalog Number SPIV2
Doc. Part No. 100020219 Pub. No. MAN0008380 Rev. A.0
Technology
Species
Sample matrix
Sample type
Protocol
Double-well indirect ELISA
– Double-strip plates
Bovine Serum Individual Short incubation
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Safety Data Sheets (SDSs) are available from thermofisher.com/support.
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General information
The PI3 virus (Parainfluenza type 3 virus) is a Parainfluenza virus responsible for many respiratory and
digestive illnesses in bovine.
Generally, the PI3 virus is considered to be a co-responsible agent that predisposes cattle to develop
bacterial infections (Pasteurella hemolytica) and/or other viral infections (BVD, IBR…). The transmission
essentially takes place by nasal discharge, which is facilitated during animal transport (direct contact,
absence of ventilation, confined conditions). Classic symptoms are hyperthermia, cough, oculonasal
discharge, anorexia, dyspnea, and diarrhea. The PI3 virus also has immunosuppressive activity, which
predisposes the animals to secondary infections.
It is strongly recommended to diagnose PI3 virus by the detection of seroconversion.
Procedure overview
The kit is based on the principle of a double-well indirect ELISA.
1. Samples and controls are added to the viral PI3 antigen (AgV, odd columns) and control antigen
(AgC, even columns) coated microplate. If present, anti-PI3 virus specific antibodies bind to the AgV
and form antigen-antibody complexes. Non-specific antibodies bind to the AgC.
2. After washing, an anti-bovine HRP-labelled conjugate is added, binding to the antibodies previously
attached to the plate.
3. Unbound conjugate is removed by washing, followed by addition of a chromogenic substrate. A blue
color results from substrate oxidation by the HRP-conjugate.
4. After stopping the reaction, the color turns to yellow. Results are read by an ELISA plate reader.
Yellow color indicates the presence of antibodies in the sample. A non-specific reaction results in
equal intensity of yellow color in AgV and AgC wells. A specific reaction results in higher intensity
color in the AgV well compared to the corresponding AgC well. The difference between the two wells
is proportional to the amount of specific antibodies in the sample.
2 PrioCHECK™ Bovine PI3 Ab Serum Kit Instructions for Use
Kit contents and storage
Component Description
Quantity
(96 tests)
Storage
1 - Coated microplate PI3
6 double-strips of 16 wells
2 units
5±3°C
(1)
2 - Negative C. PI3
Negative Control PI3 (NC)
500 µL
5±3°C
3 - Positive C. PI3
Positive Control PI3 (PC)
500 µL
4 - Conjugate (100x) PI3
Anti-bovine HRP labeled conjugate, 100X concentrate
350 µL
5±3°C
(2)
A - Wash (10x)
Wash solution, 10X concentrate
125 mL
5±3°C
B - Dilution Buffer PI3
Sample and Conjugate Dilution Buffer.
The buffer can be opaque, which does not affect the
performance of the kit.
50 mL
C - Substrate
Substrate solution
24 mL
D - Stop
Stop solution
24 mL
Adhesive plate covers
4
RT
(3)
(1) Unused strips should be stored within the enclosed sealed plastic bag with a dessicant bag at 5±3°C.
(2) The diluted conjugate solution should be used immediately after preparation.
(3) Room temperature
Materials required but not provided
Unless otherwise indicated, all materials are available through thermofisher.com.
Single and multichannel micropipettes
Disposable pipette tips
Distilled or deionized water
Microplate incubator (37±2°C)
Disposable containers
ELISA reader with 450-nm filter or 450- and 620-nm filters
Important procedural guidelines
• Do not mix reagents from different kit batches.
• Avoid contaminating the reagents by using single-use sampling equipment.
• Do not pipette reagents by mouth.
Prepare samples
Fresh, refrigerated (8 days at 5±3°C) or previously frozen (1 year at < −16°C) serum can be used.
Previously homogenized individual sera and controls are tested at a 1:10 dilution.
NOTE: Use of an internal reference is recommended for each test series. An internal reference
(Cat. No. RPI3) is available.
Prepare reagents
• Reagents 1 - Coated microplate PI3, B - Dilution Buffer PI3, C - Substrate, and D - Stop are ready
for use.
• Reagents 2 - Negative C. PI3 and 3 - Positive C. PI3 are tested as samples.
• Dilute A - Wash (10x) solution 1:10 in distilled/deionized water.
Example: for 1 strip, dilute 2 mL of A - Wash (10x) solution in 18 mL of water; for 1 plate, dilute
25 mL of A - Wash (10x) solution in 225 mL of water.
Mix after dilution. The diluted Wash solution can be stored 1 month at 5±3°C.
NOTE: Due to the high salt concentration, crystals may form in A - Wash (10x) solution. Prior to
dilution, shake the bottle to dissolve any crystals.
• Dilute 4 - Conjugate (100x) PI3 1:100 in B - Dilution Buffer PI3.
Mix after dilution. Use diluted Conjugate PI3 solution immediately after dilution.
PrioCHECK™ Bovine PI3 Ab Serum Kit Instructions for Use 3
Perform the ELISA procedure
NOTE: Bring the reagents to room temperature (21±4°C) before use. The tolerance for incubation times
is ±10%. Use disposable containers for distribution of reagents.
1. Distribution of controls and samples
• Add 10 µL of 2 - Negative C. PI3 solution to wells A1, A2 and B1, B2 (for example).
• Add 10 µL of 3 - Positive C. PI3 solution to wells C1, C2 and D1, D2 (for example).
• Add 10 µL of test sample per well to paired wells (one AgV well and the adjacent AgC well).
• Add 90 µL of B - Dilution Buffer PI3 reagent to each well containing controls and samples.
Gently shake the plate, and cover the plate with an adhesive plate cover. Incubate the plate for 1 hour
at 37±2°C.
2. Washing steps (4 washes)
Empty the plate and wash 4 times with 300 µL of diluted Wash solution (see “Prepare reagents”) per
well. Empty and tap the plate hard on absorbent paper to remove any traces of liquid. Washes can be
performed manually or automated using a plate washer. Do not allow the plate to dry out.
3. Conjugate distribution
Add 100 µL of diluted Conjugate PI3 solution (see “Prepare reagents”) to each well. Gently shake the
plate, and cover the plate with a new adhesive plate cover. Incubate the plate for 1 hour at 37±2°C.
4. Washing steps (4 washes)
Repeat the washing steps (step 2) as described above.
5. Test development
Add 100 µL of C - Substrate solution to each well. Gently shake the plate for 2 seconds. Incubate for
10 minutes at room temperature (21±4°C) in darkness. Do not cover the plate.
Add 100 µL of D - Stop solution to each well and in the same order as C - Substrate. Gently shake the
plate for 2 seconds.
6. Reading
Wipe the bottom of the plates with a soft tissue to remove any dust. Read the plate within 30 minutes
after stopping the reaction at 450 nm or at dual wavelengths of 450–620 nm on a microplate reader.
Calculation
For each control and sample, calculate the corrected Optical Density (ODc) by subtracting the
AgC well OD (OD AgC) from the AgV well OD (OD AgV):
ODc = OD AgV − OD AgC
NOTE: For negative samples, ODc may be < 0.
Calculate the mean ODc of the positive control (ODc m PC) and of the negative control (ODc m NC).
For each sample, calculate the sample/positive ratio (S/P):
S/P = ODc Sample / ODc m PC
Validation
The test is validated if:
ODc m PC > 0.600 and ODc m NC < 0.150
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9 December 2016
Interpretation of results
Result
Interpretation
S/P ≤ 0.10
Negative
0.10 < S/P < 0.20
Doubtful
0.20 ≤ S/P < 0.40
+ Positive
0.40 ≤ S/P < 0.60
++ Positive
0.60 ≤ S/P < 0.80
+++ Positive
S/P ≥ 0.80
++++ Positive
Animals with a “doubtful” result should be
retested.
Seroconversion diagnosis
Sample collection and distribution
To determine seroconversion (significant variation
of anti-PI3 antibody titre between early and late
blood samples):
• Use paired samples (early serum, late serum)
from the same animal.
• Test sample pairs on the same plate.
• Use the kit procedure.
Interpretation of results
For each pair of samples, calculate the following
parameters:
• ODc of early sample (ODc EAR)
• ODc of late sample (ODc LAT)
• Ratio S/P of early sample:
S/P EAR = ODc EAR / ODc m PC
• Ratio S/P of late sample:
S/P LAT = ODc LAT / ODc m PC
• Index Conversion (IC) of sample pair:
IC = ODc LAT / ODc EAR
Seroconversion is indicated by:
S/P LAT ≥ 0.20 and IC ≥ 2
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Customer and technical support
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manufacturer.
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The information in this guide is subject to change without notice.
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Revision history: Pub. No. MAN0008380 (English)
Rev.
Date
Description
A.0 9 December 2016
Updated to the current document
template, with associated updates to
the warranty, trademarks, and logos.
1.0
12 July 2013
Baseline for revision history
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only to perform veterinary diagnostic services including reporting
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