Thermo Fisher Scientific PrioCHECK Bovine PI3 Ab Serum Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: PrioCHECK Bovine PI3 Ab Serum Kit

Type: Operating instructions

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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Wear appropriate protective eyewear, clothing, and gloves.

Safety Data Sheets (SDSs) are available from thermofisher.com/support.

WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this

product’s page at thermofisher.com. Wear appropriate protective eyewear, clothing, and gloves.

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

(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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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

Documentation and support

Customer and technical support

Technical support: visit

thermofisher.com/askaquestion

Visit thermofisher.com/support for the latest in

services and support, including:

• Worldwide contact telephone numbers

• Order and web support

• User guides, manuals, and protocols

• Certificates of Analysis

• Safety Data Sheets (SDSs; also known as

MSDSs)

NOTE: For SDSs for reagents and chemicals

from other manufacturers, contact the

manufacturer.

Limited product warranty

Life Technologies Corporation and/or its

affiliate(s) warrant their products as set forth in the

Life Technologies' General Terms and Conditions

of Sale found on Life Technologies' website at

www.thermofisher.com/us/en/home/global/

terms-and-conditions.html. If you have any

questions, please contact Life Technologies at

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8211 AR Lelystad | The Netherlands

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE

TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE

FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR

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FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

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

Limited Use Label License No. 473: Veterinary Diagnostic Assay.

The purchase of this product conveys to the purchaser the limited,

non-transferable right to use the purchased amount of the product

only to perform veterinary diagnostic services including reporting

the results of such services for a fee, and internal research for the

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trademarks are the property of Thermo Fisher Scientific and its

subsidiaries unless otherwise specified.

Thermo Fisher Scientific PrioCHECK Bovine PI3 Ab Serum Kit Operating instructions

Thermo Fisher Scientific PrioCHECK Bovine PI3 Ab Serum Kit Operating instructions

Thermo Fisher Scientific PrioCHECK Bovine PI3 Ab Serum Kit Operating instructions

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