Thermo Fisher Scientific PrioCHECK Bovine BVDV PI focus Ag Strip Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: PrioCHECK Bovine BVDV PI focus Ag Strip Kit

Type: Operating instructions

For Veterinary Use Only. For In Vitro Use Only.

INSTRUCTIONS FOR USE

PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit

ELISA for

in vitro

detection of Bovine Viral Diarrhea Virus antigen in ear notch tissue of cattle

Catalog Number 7610140

Pub. No. MAN0013846 Rev. A.0

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.

Introduction

The bovine virus diarrhea virus (BVDV) infections cause large annual economic losses to the cattle industry. Additionally BVDV has also got a negative

impact on animal welfare.

The Applied Biosystems™ PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit is specifically designed to detect persistently infected (PI) animals with a very

high specificity. The test detects different subtypes of BVDV (Ia, Ib, and II) antigen in ear tissue samples originating from cattle.

The PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit test can be used to diagnose persistent infections of BVDV in individual animals. Moreover, the test is

a valuable tool in large-scale screening and eradication programs.

Validation data show that by the presence of maternal antibodies in the ear sample tissue the Ag-result can be influenced (shielding). As a consequence

ELISA-tests can show an incomplete sensitivity for persistently infected (PI) animals. Therefore an additional control for the presence of BVDV antibodies in

ear sample tissue of animals under the influence of maternal antibodies is recommended.

Test principle

The PrioCHECK™ Bovine BVDV PI focus Ag

Strip Kit ELISA employs two monoclonal

antibodies (mAb’s). The two mAb’s recognize

different epitopes located on the highly

conserved NS-3 non-structural protein (p80)

of BVDV. One mAb is coated to the plate and

the second mAb is used as a conjugate. For

antigen detection undiluted ear tissue

samples are simultaneously incubated with

buffer in the wells of the coated plate. This

incubation step is followed by a washing

step. Subsequently, the working dilution of

the Horse radish peroxidase conjugate is

dispensed and after an incubation period a

second washing step follows. In the subsequent step Chromogen (TMB) Substrate is dispensed to the wells

and after incubation the color development is stopped and the optical density at 450 nm is measured.

During the first incubation period BVDV antigens present in the sample will bind to the coated BVDV

specific monoclonal antibodies. Subsequently, the plates are washed and incubated with a Horse radish

peroxidase conjugated BVDV-specific monoclonal antibodies (mAb against another epitope of NS-3).

Finally, the bound Conjugate and therefore BVDV-antigen from the test sample is visualized by adding a

Chromogen (TMB) Substrate. In the case of a positive sample color development will be visible. In case of

a negative sample there will be no color development.

Kit components

10 plate kit for 900 samples. 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.

Component

Description

1: Test Plate

Ten strip Test Plates.

2: Extraction Buffer

Ready-to-use. Four vials, each containing 60 mL Extraction Buffer.

3: Extraction Buffer Additive

Lyophilized. Four vials, each vial contains Extraction Buffer Additive for one vial

of Extraction Buffer. Shelf life of Extraction Buffer Solution: 1 month at 5±3°C.

4: Conjugate (30x)

Two vials, each vial contains 2.5 mL Conjugate. Diluted conjugate not stable,

prepare just before use.

5: Conjugate Buffer

Ready-to-use. Two vials, each vial contains 60 mL Conjugate Buffer.

6: Dilution Buffer

Ready-to-use. Two vials, each vial contains 60 mL Dilution Buffer.

7: Washing Fluid (200x)

200x concentrated, dilute before use. One vial contains 60 mL Washing

Fluid. Shelf life of washing solution: 1 week at 22±3°C.

8: Reference 1

Lyophilized. One vial contains 2.0 mL Reference 1. Shelf life of reconstituted

reference 1: until expiry date at −20°C.

9: Reference 2

Lyophilized. One vial contains 2.0 mL Reference 2. Shelf life of reconstituted

reference 2: until expiry date at −20°C.

10: Reference 3

Lyophilized. One vial contains 2.0 mL Reference 3. Shelf life of reconstituted

reference 3: until expiry date at −20°C.

11: Chromogen (TMB) Substrate

Ready-to-use. Two vials, each vial contains 60 mL Chromogen (TMB)

Substrate.

12: Stop Solution

Ready-to-use. Two vials, each vial contains 60 mL Stop Solution.

Additional kit contents

• Package Insert

• 20 plate sealers to cover the strips during incubation

Additional material required

Use

Description

(1)

General

Laboratory equipment according to national safety regulations.

Analysis of

results

Plate Reader. The reader has to have an appropriate filter set to read the plates at 450 nm.

Optional

Plate washer. Liquid handler.

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

2 PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit Instructions for Use

Test procedure

Precautions

• National Safety Regulations must be strictly followed.

• The PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit 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.

Notes

To achieve optimal results with the PrioCHECK™ Bovine BVDV PI focus

Ag Strip Kit, the following aspects must be considered:

• The Test Procedure protocol must be strictly followed.

• All reagents of the kit must be equilibrated to room temperature

(22±3°C) before use.

• Pipette tips have to be changed for every pipetting step.

• Separate solution reservoirs must be used for each reagent.

• Kit components must not be used after their expiry date or if changes in

their appearance are observed.

• Kit components of different kit lot numbers must not be used together.

• Demineralized or water of equal quality must be used for the test.

• Only fresh ear notch tissue samples that have been well preserved by

desiccation should be tested. Macerated tissue should not be used for

testing.

Solutions to be made in advance for extraction of the test samples

Extraction buffer solution

Equilibrate the vials to 22±3°C and reconstitute the Extraction Buffer

Additive (Component 3) with Extraction Buffer (Component 2) and add the

complete content of one vial extraction buffer additive to one vial of

Extraction Buffer.

Stability of Extraction Buffer solution: 1 month at 5±3°C.

Preparatory steps

1. Transfer the ear notch sample from the sample container into a deep

well plate.

2. Add 200 µL of extraction buffer solution to the deep well plate.

3. Incubate overnight (12 to 18 hours) at 22±3°C.

Solutions to be made in advance

Conjugate dilution

Dilute Conjugate (30x) (Component 4) 1:30 in Conjugate Buffer

(Component 5); e.g. for one plate prepare 12 mL (add 400 µL Conjugate

(30x) to 11.6 mL Conjugate Buffer).

Note: The diluted conjugate must be prepared just before use.

Washing solution

Dilute Washing Fluid (200x) (Component 7) 1:200 in demineralized water.

The amount of Washing Fluid is sufficient to prepare a final volume of

12 liters washing solution.

Stability of washing solution: 1 week stored at 22±3°C.

Remark: Commercial available ELISA washers can be used. If not available,

washing of the Test Plates can be done by dispensing at least 200 µL of

washing solution to all wells of the Test Plate. Subsequently, empty the Test

Plate and repeat as many times as prescribed. It is not necessary to soak the

Test Plate between washings. Tap the Test Plate firmly after the last

washing step.

Reference 1–3

Equilibrate the vials to 22±3°C and reconstitute the Reference 1–3

(Component 8–10) with 2.0 mL Dilution Buffer (Component 6). The

reconstituted references can be stored in portions of 150 µL at −20°C until

expiry date.

Reconstitution of the lyophilized material should be performed as follows:

1. Equilibrate the vial to 22±3°C.

2. With the vial in an upright position, tap the vial gently against the

worktop to ensure that the content is on the bottom of the vial.

3. Carefully open the vial.

4. Add the specified amount of Dilution Buffer (Component 7).

5. Replace the stopper on the vial and allow the lyophilized material to

dissolve.

6. Gently agitate the vial so that any remaining dry material will be

dissolved.

7. Allow the material to stand at least for 15 minutes at 22±3°C before use.

8. Mix gently and intermittently (formation of foam should be avoided).

Incubation of test samples

Antigen detection:

1. Label each strip of the Test Plate (Component 1) with a marker pen.

2. Dispense 50 µL of Dilution Buffer (Component 6) to all wells.

3. Dispense 50 µL of Reference 1 to the wells A1 and B1 (= OD max).

4. Dispense 50 µL of Reference 2 to the wells C1 and D1.

5. Dispense 50 µL of Reference 3 to the wells E1 and F1 (= OD blank).

6. Dispense 50 µL of the test samples into the remaining wells.

7. Seal the Test Plate and shake it gentle.

8. Incubate for 60±10 minutes at 22±3°C.

Incubation with conjugate

1. Empty the Test Plate and wash it 6 times with washing solution. Tap

the plate firmly after the last wash cycle.

2. Dispense 100 µL of diluted conjugate to all wells.

3. Seal the Test Plate.

4. Incubate the Test Plate for 60±10 minutes at 22±3°C.

Incubation with Chromogen (TMB) Substrate

1. Empty the Test Plate and wash it 6 times with washing solution. Tap

the plate firmly after the last wash cycle.

2. Dispense 100 µL of the Chromogen (TMB) Substrate (Component 11) to

all wells.

3. Incubate the Test Plate for 15 to 25 minutes at 22±3°C.

4. Add 100 µL of Stop Solution (Component 12) to all wells.

5. Mix the contents of the wells of the Test Plate.

Note: Start the addition of Stop Solution 15 to 25 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

Measure the optical density (OD) of the wells at 450 nm within 20 minutes

after color development has been stopped.

Calculate the mean OD450 of wells E1 and F1 (= OD blank).

Calculate the corrected OD450 of all samples by subtracting the OD blank.

Calculate the mean of the corrected OD450 of wells A1 and B1

(= corrected OD max).

Calculate the percent positivity (PP) of Reference 2 and of the test samples

according to the formula below.

Note: The corrected OD450 of all samples is expressed as percent positivity

(PP) relative to the corrected mean OD450 of wells A1 and B1

(= corrected OD max).

PP = (corrected OD450 test sample / corrected ODmax) × 100%

Result interpretation

Validation criteria BVDV-Ag test

1. The mean OD450 of Reference 3 (= OD blank) must be ≤0.300.

2. The corrected OD450 of Reference 1 must be ≥0.800.

3. The percent positivity of Reference 2 must be <30%.

Not meeting these criteria is reason to discard the results of that specific test

plate.

Note: If the corrected OD max is below 0.800 possibly the Chromogen

(TMB) Substrate is too cold. In that case prewarm the solution to 22±3°C or

incubate up to 30 minutes.

Interpretation of BVDV-Ag test

PP = ≥20%

Positive

BVDV-antigen present in the test sample.

PP = <20%

Negative

No detectable antigen present in the test sample.

Note: PrioCHECK™ Bovine BVDV PI focus Ag Strip Kit is a highly specific

test for the detection of PI-animals. However, if there is any doubt about the

status of a valuable, positive live animal, retest another sample collected

2–3 weeks (ELISA) or 6 weeks (PCR/NASBA) after the initial sample was

collected to confirm persistent infection.

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

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Revision history of Pub. No. MAN0013846 (English)

Rev.

Date

Description

A.0 1 October 2019

New document. Converted the legacy document (PrioCHECK BVDV Ag

PIfocus 10plate_v2.1_e.doc) to the current document template, with

associated updates to the publication number, limited license

information, warranty, trademarks, and logos.

Important Licensing Information: These products may be covered by one or more Limited Use Label

Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use

Label Licenses.

Fisher Scientific and its subsidiaries unless otherwise specified.

Thermo Fisher Scientific PrioCHECK Bovine BVDV PI focus Ag Strip Kit Operating instructions

Thermo Fisher Scientific PrioCHECK Bovine BVDV PI focus Ag Strip Kit Operating instructions

Thermo Fisher Scientific PrioCHECK Bovine BVDV PI focus Ag Strip Kit Operating instructions

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