Thermo Fisher Scientific PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit

Type: Operating instructions

INSTRUCTIONS FOR USE

For Veterinary Use Only. For In Vitro Use Only.

PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit

Immunoenzymatic test for specific detection of anti-p80 BVDV and BDV antibodies in

ruminant serum.

Catalog Number BVDI5

Doc. Part No. 100020235 Pub. No. MAN0009388 Rev. A.0

Technology

Species

Sample matrix

Test type

Protocol

Single-well blocking ELISA

-Strip plates

Bovine

Ovine Serum Individual

Short incubation

Long incubation

Pool

Long incubation

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions.

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

Bovine Viral Diarrhea Virus (BVDV) is a virus belonging to the Pestivirus genus of the Flaviviridae family.

Two other animal viruses belong to the same Pestivirus genus: Border Disease Virus (BDV) in sheep and

Classic Swine Fever (CSF) in pigs.

BVDV is one of the most widespread pathogenic viruses in livestock, causing significant losses in the milk

and meat industries worldwide.

The virus crosses the placenta of infected gestating cows, causing reproductive losses due to abortions,

still-born calves, and calves that die early. Some surviving calves are immunotolerant to the virus, and

these animals excrete large quantities of infectious virus for the rest of their lives. It is important to

identify these carrier animals to break the cycle of infection in the herd. Carrier animals often die of

mucosal disease in their first two years of life.

Diagnosis of BVDV through detection of specific antibodies has a number of applications:

•Detection of viral circulation in livestock

•Pre-detection of immunotolerant, persistently infected animals (PI)

•Monitoring of herds after elimination of the PI

•Implementation of epidemiological investigations

The Applied Biosystems™ PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit can be used on the

following samples: individual and pooled bovine and ovine sera to detect anti-BVDV and anti-BDV

antibodies.

Procedure overview

The test is based on the principle of a Blocking ELISA (competition assay).

1. The samples and controls are added to the BVD/BD antigen (Ag)-coated plate. Any specific BVD/BD

antibodies bind to the Ag.

2. After washing, a conjugate (anti-p80 monoclonal antibody) labeled with peroxidase is added, binding

to the unbound p80 that remains in the plate.

3. The unbound conjugate is eliminated by washing before the addition of a chromogenic substrate. A

blue color results from substrate oxidation by the peroxidase in the conjugate.

4. After stopping the reaction, the color turns yellow. The results are read by an ELISA plate reader.

Positive samples produce relatively weak to no color.

2 PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit Instructions for Use

Kit contents and storage

Component Description

Quantity

(480 tests)

Storage

1 - Coated microplate BVD

BVD-coated microplate, 12 strips of 8 wells

5 units

5±3°C

(1)

2a - Serum Negative C. BVD

BVD negative control (NC)

1 mL

5±3°C

3a - Serum Positive C. BVD

BVD positive control (PC)

1 mL

4 - Conjugate BVD

Conjugate BVD, anti-p80 HRP (pink)

60 mL

A - Wash (10x)

Wash solution, 10x concentrate

250 mL

B - Sample DB BVD

BVD sample dilution buffer (green)

60 mL

C - Substrate

Substrate solution

60 mL

D - Stop

Stop solution

60 mL

Adhesive plate covers

(2)

(1) Unused strips can be stored in the sealed pouch with desiccant (supplied with the kit) at 5±3°C until the kit’s expiry date.

(2) Room temperature

Materials required but not provided

Unless otherwise indicated, all materials are available through thermofisher.com.

Single- and multi-channel micropipettes

Disposable pipette tips

Distilled or deionized water

Microplate incubator (37±2°C)

Disposable containers

ELISA reader equipped with a 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.

Preparation of samples

Fresh, refrigerated serum (8 days at 5±3°C) or frozen serum (1 year at < −16°C) can be used.

The samples are homogenized in advance.

•Individual sera and controls are tested at a 1:10 dilution (short or long incubation).

•Pooled sera from a group of animals raised on the same farm (long incubation only):

-For epidemiological use aimed at evaluating positive prevalence in homogeneous groups of

animals (pool of 2 to 20 sera), the pooled sera and controls are tested at a 1:20 dilution.

-For diagnostic use aimed at detecting at least one seropositive animal in a pool of 2 to 10 sera, the

pooled sera and controls are tested at a 1:2 dilution.

NOTE: The use of an internal tracer is highly recommended for each test series. An internal reference

(Cat. No. RBVD) is available.

Preparation of reagents

•Reagents 1 - Coated microplate BVD, B - Sample DB BVD, 4 - Conjugate BVD, C - Substrate and

D - Stop are ready for use.

•Reagents 2a - Serum Negative C. BVD and 3a - Serum Positive C. BVD should be tested as samples.

•A - Wash (10x) solution should be diluted 1:10 in distilled/deionized water.

Example: for one strip: 2 mL of A - Wash (10x) solution in 18 mL of water; for one plate: 25 mL of

A - Wash (10x) solution in 225 mL of water.

Mix after diluting. The diluted Wash solution can be stored for 1 month at 5±3°C.

NOTE: Due to the high salt concentration, crystals may form in the A - Wash (10x) solution. Shake the

bottle to dissolve any crystals prior to dilution.

PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit Instructions for Use 3

Perform the ELISA test

NOTE: Bring the reagents to room temperature (21±4°C) before performing the test. The tolerance range

for incubation times is ±10%. The use of disposable containers is recommended for distribution of

reagents.

1. Distribution of samples and controls

A. Individual serum samples (at a 1:10 dilution):

•Add 10 µL of reagent 3a - Serum Positive C. BVD to wells A1 and B1 (for example).

•Add 10 µL of reagent 2a - Serum Negative C. BVD to wells C1 and D1 (for example).

•Add 10 µL of the serum to be analyzed to the following wells.

•Add 90 µL of reagent B - Sample DB BVD to each well containing the samples and controls.

B1. Pooled serum samples - Epidemiological use (at a 1:20 dilution):

•Add 5 µL of reagent 3a - Serum Positive C. BVD to wells A1 and B1 (for example).

•Add 5 µL of reagent 2a - Serum Negative C. BVD to wells C1 and D1 (for example).

•Add 5 µL of the pooled serum to be analyzed to the following wells.

•Add 95 µL of reagent B - Sample DB BVD to each well containing the samples and controls.

B2. Pooled serum samples - Diagnostic use (at a 1:2 dilution):

•Add 50 µL of reagent 3a - Serum Positive C. BVD to wells A1 and B1 (for example).

•Add 50 µL of reagent 2a - Serum Negative C. BVD to wells C1 and D1 (for example).

•Add 50 µL of the pooled serum to be analyzed to the following wells.

•Add 50 µL of reagent B - Sample DB BVD to each well containing the samples and controls.

Gently shake the plate, then cover it with an adhesive plate cover.

Individual serum analysis: Incubate the plate overnight (16 to 18 hours) at 5±3°C or 1 hour at 37±2°C.

NOTE: The kit has better detectability with long incubation.

Pooled sera analysis: Incubate the plate overnight (16 to 18 hours) at 5±3°C

2. Washing steps

Empty the plate and perform 4 washes with the diluted Wash solution (see “Preparation of reagents”)

using 300 µL per well. Empty and tap the plate on absorbent paper to eliminate any traces of liquid.

Washes can be performed either manually or automated using a plate washer. Do not allow the plate to

dry out.

3. Conjugate distribution

Add 100 µL of reagent 4 - Conjugate BVD to each well. Gently shake the plate, and cover the plate using a

new adhesive plate cover. Incubate the plate for 1 hour at 37±2°C.

4. Washing steps

Repeat the “Washing steps” as described above.

5. Test development

Add 100 µL of solution C - Substrate to each well. Gently shake the plate for 2 seconds. Incubate for

10 minutes at room temperature (21±4°C) and in darkness. Do not cover the plate.

Add 100 µL of solution D - Stop to each well and in the same order as solution C - Substrate. Gently

shake the plate for 2 seconds.

6. Reading

Dry the bottom of the plates with a soft tissue to remove any dust. Read the plate within 30 minutes of

stopping the reaction at 450 nm (monochromatic) or at 450–620 nm (bichromatic).

Calculation

Calculate the average OD of the PC (ODm PC), and that of the NC (ODm NC).

For each sample, calculate the percent inhibition (% Inh):

% Inh = [ (ODm NC − OD Sample) / ODm NC ] × 100

NOTE: For negative samples, % Inh may be < 0.

thermofisher.com/support | thermofisher.com/askaquestion

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4 April 2017

Validation

The test is validated if:

% Inh PC > 60% and ODm NC > 0.650

Interpretation of results

Table 1 Individual serum

Results

Quantitative interpretation

Qualitative interpretation

% Inh < 50

–

Negative

50 ≤ % Inh < 80

Weak positive

% Inh ≥ 80

Strong positive

Table 2 Pool of 2 to 20 sera - Protocol 1:20 - Epidemiological use

Results

% Seroprevalence

Interpretation

% Inh < 65

The positive prevalence of anti-p80

antibodies is low, generally < 50%

No or low risk of viral circulation

% Inh ≥ 65

The positive prevalence of anti-p80

antibodies is high, generally > 50%

Suspicion of viral passage. High risk of

presence of PI or transient viremic animals

Table 3 Pool of 2 to 10 sera - Protocol 1:2 - Diagnostic use

Results

Qualitative interpretation

Status

% Inh < 30

Negative

Not infected

% Inh ≥ 30

Positive

Recently infected or infected

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

thermofisher.com/support.

Prionics Lelystad B.V. | Platinastraat 33 | 8211 AR

Lelystad | The Netherlands

Translated from the French Pub. No. MAN0007704 Rev. A.0.

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

CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING

FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0009388 (English)

Rev.

Date

Description

A.0 4 April 2017

Updated to the current document

template, with associated updates to

the warranty, trademarks, and logos.

1.0

4 October 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

sole benefit of the purchaser. No right to resell, repackage, or

distribute this product, or any of its components, is conveyed ex-

pressly, by implication, or by estoppel. For information on obtaining

additional rights, please contact outlicensing@lifetech.com or

Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad,

California 92008.

trademarks are the property of Thermo Fisher Scientific and its

subsidiaries unless otherwise specified.

Thermo Fisher Scientific PrioCHECK™ Ruminant BVD & BD p80 Ab Serum Kit Operating instructions

Thermo Fisher Scientific PrioCHECK&trade; Ruminant BVD & BD p80 Ab Serum Kit Operating instructions

Thermo Fisher Scientific PrioCHECK&trade; Ruminant BVD & BD p80 Ab Serum Kit Operating instructions

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