Thermo Fisher Scientific PrioCHECK Calf Operating instructions

Brand: Thermo Fisher Scientific

Model: PrioCHECK Calf

Type: Operating instructions

INSTRUCTIONS FOR USE

For Veterinary Use Only. For In Vitro Use Only.

PrioCHECK™ Calf Enteritis Ag Feces Kit

Immunoenzymatic test for simultaneously detecting the antigens Rotavirus (Group A),

Coronavirus, E. coli K99, and Cryptosporidium parvum in ruminant feces

Catalog Number LSIDTT1

Doc. Part No. 100020263 Pub. No. MAN0008594 Rev. B.0

Technology

Species

Sample matrix

Test type

Protocol

Double-well Antigen-capture

ELISA Strip plates

Ruminants Feces Individual Short 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

Diarrhea constitutes one of the leading causes of death in calves and all young ruminants. Bovine neonatal

gastroenteritis is a multifactor disease. It may be induced by a virus (Rotavirus and Coronavirus), bacteria

(Salmonella, Enterotoxigenic Colibacillus) or protozoans (Cryptosporidium parvum). Laboratory tests are

obligatory for determining causes of diarrhea since identifying the causative agent based on clinical

symptoms is impossible.

Procedure overview

The test is based on the principle of an Antigen-capture ELISA.

1. The samples and controls are deposited in the plate coated with anti-pathogenic antibodies (AbP) and

control antibodies (AbC). The antigens, including specific antigens of the potentially present

pathogens being investigated, bind to the AbP while only the non-specific Ag bind to the AbC.

2. After washing, specific monoclonal conjugates labeled with peroxidase are added, binding to the

antigens previously attached to the microwells.

3. The unbound conjugates are 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.

The appearance of a yellow coloration indicates the presence of antigens. A non-specific reaction

results in an equivalent coloration between the AbP and AbC wells. A specific reaction results in a

more significant coloration in the AbP well compared to the AbC well. The difference between these

two colorations indicates the level of positivity in the sample.

2 PrioCHECK™ Calf Enteritis Ag Feces Kit Instructions for Use

Kit contents and storage

Component Description

Quantity

(12 tests)

Storage

1 - Coated microplate DTT

DTT-coated microplate, 12 strips of 8 wells:

• Line A: specific anti-

Rotavirus

group A antibody

• Line B: control antibody

• Line C: specific anti-

Coronavirus

antibody

• Line D: control antibody

• Line E: specific anti-

E. coli

K99 antibody

• Line F: control antibody

• Line G: specific anti-

Cryptosporidium

antibody

•

Line H: control antibody

1 unit 5±3°C(1)

3a - Positive C.

Rotavirus

positive control (red)

1.4 mL

5±3°C

3b - Positive C.

Coronavirus

positive control (yellow)

1.4 mL

3c - Positive C.

E. coli

K99

E. coli

K99 positive control (blue)

1.4 mL

3d - Positive C.

Cryptosporidium

positive control (green)

1.4 mL

4a - Conjugate

Rotavirus

HRP conjugate (red)

3.5 mL

4b - Conjugate

Coronavirus

HRP conjugate (yellow)

3.5 mL

4c - Conjugate

E. coli

K99

E. coli

K99 HRP conjugate (blue)

3.5 mL

4d - Conjugate

Cryptosporidium

HRP conjugate (green)

3.5 mL

A - Wash (10x)

Wash solution, 10X concentrate

60 mL

B - Sample DB (5x) DTT

Sample dilution buffer (5X) DTT, 5X concentrate

25 mL

C - Substrate

Substrate solution

12 mL

D - Stop

Stop solution

12 mL

Adhesive plate covers

RT(2)

Plastic caps

6 × 8

(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.

Disposable pipette tips

Single- and multi-channel micropipettes

Distilled or deionized water

Disposable containers

Hemolysis tubes

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.

Information on samples

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

Dilute the feces to 1:2 for the test.

Prepare reagents

• The reagents 1 - Coated microplate DTT, 3a - Positive C. Rotavirus, 3b - Positive C. Coronavirus,

3c - Positive C. E. coli K99, 3d - Positive C. Cryptosporidium, 4a - Conjugate Rotavirus,

4b - Conjugate Coronavirus, 4c - Conjugate E. coli K99, 4d - Conjugate Cryptosporidium,

C - Substrate, and D - Stop are ready to use.

• Dilute the reagent B - Sample DB (5x) DTT 1:5 in distilled/deionized water.

Example: dilute 10 mL of the reagent B - Sample DB (5x) DTT in 40 mL of water.

Mix after diluting. The diluted solution Sample DB DTT may be stored at 5±3°C for use within

8 days.

PrioCHECK™ Calf Enteritis Ag Feces Kit Instructions for Use 3

• 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. The

crystals can be dissolved by shaking. The solution should be mixed thoroughly prior to dilution.

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

components.

If one or more samples are not tested using all of the pathogens, cover all wells containing unused

pathogens with plastic caps.

1. Preparation of samples

Dilute the fecal matter 1:2 in the diluted solution Sample DB DTT (see “Prepare reagents”). We

recommend diluting 500 µL of feces in 500 µL of the diluted solution Sample DB DTT using a hemolysis

tube. If the sample consistency renders volume measurement or mixing difficult, add a volume of the

diluted solution Sample DB DTT equal to the sample volume into a hemolysis tube and vigorously shake

together in the vortex. Numerous agitation cycles may be required. Do not centrifuge.

2. Distribution of samples and controls

Test the positive controls without additives or dilution, adding 100 µL of:

• 3a - Positive C. Rotavirus: wells A1 and B1

• 3b - Positive C. Coronavirus: wells C1 and D1

• 3c - Positive C. E. coli K99: wells E1 and F1

• 3d - Positive C. Cryptosporidium: wells G1 and H1

NOTE: The positive control 3d - Positive C. Cryptosporidium may contain aggregates, thus making it

harder to pipette. In this case, we recommend that you vortex the tube well and cut the cone tip to sample

the control.

Add 100 µL of the diluted samples to each well according to the following layout: sample 1 in the

column 2 wells, sample 2 in the column 3 wells, etc.

Gently shake the plate, then cover it with an adhesive plate cover. Incubate the plate for 1 hour at 21±4°C.

3. Washing steps

Empty the plate and perform 4 washes with the diluted Wash solution (see “Prepare 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 using an automatic washer. Do not allow the plate to dry out.

4. Distribution of conjugate

Add 100 µL of the conjugates to each well, according to the following layout:

• 4a - Conjugate Rotavirus: lines A and B

• 4b - Conjugate Coronavirus: lines C and D

• 4c - Conjugate E. coli K99: lines E and F

• 4d - Conjugate Cryptosporidium: lines G and H

Gently shake the plate, and cover the plate using a new adhesive plate cover. Incubate the plate for

1 hour at 21±4°C.

5. Washing steps

Repeat the step outlined above in “3. Washing steps.”

6. 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.

7. 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 dual wavelengths of 450 and 620 nm.

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

13 October 2016

Calculation

Calculate the corrected Optical Density (ODc) for

each sample and pathogen by deducting the OD

for the corresponding control well from the OD

obtained on each specific well.

ODc = OD AbP – OD AbC

Carry out the same operation for the Antigen

Positive Controls (PC).

NOTE: For negative samples, ODc may be < 0.

Validation

The test is validated if each positive control

reaches: ODc ≥ 0.400.

Interpretation of results

The interpretation of results for the 4 pathogens is

as follows:

Results

Interpretation

ODc ≥ 0.150

Positive

< 0.150

Negative

Example (For 2 samples using 3 strips):

ODc

Interpretation

Pathogens

Samp1

Samp2

Samp1

Samp2

Samp1

Samp2

2.269

2.237

0.184

2.139

2.055

0.032 Positive

Validated

Pos

Neg

Rotavirus

0.130

0.182

0.152

1.865

0.090

1.567

1.798 0.008

1.469

Positive

Validated

Neg

Pos

Coronavirus

0.067

0.082

0.098

2.352

0.260

0.067

2.250

0.170

−0.005

Positive

Validated

Pos

Neg

E. Coli

K99

0.102

0.090

0.072

1.955

0.212

0.320

1.874 0.012 0.060 Positive

Validated

Neg Neg

Cryptosporidium

0.081

0.200

0.260

Technical Support can provide you with an Excel file for automatically carrying out calculations and

interpretations of results.

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. MAN0008595 Rev. B.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. MAN0008594 (English)

Rev.

Date

Description

B.0 13 October 2016

Updated to the current document

template, with associated updates to

the warranty, trademarks, and logos.

A.0

22 January 2014

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

expressly, 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 Calf Operating instructions

Brand: Thermo Fisher Scientific

Model: PrioCHECK Calf

Type: Operating instructions

Download PDF

report

Thermo Fisher Scientific PrioCHECK Calf Operating instructions

Thermo Fisher Scientific PrioCHECK Calf Operating instructions

Related papers