Thermo Fisher Scientific PrioCHECK Porcine Toxoplasma Ab Kit User guide

Brand: Thermo Fisher Scientific

Model: PrioCHECK Porcine Toxoplasma Ab Kit

Type: User guide

For Veterinary Use Only. For In Vitro Use Only.

INSTRUCTIONS FOR USE

PrioCHECK™ Porcine Toxoplasma Ab Kit

ELISA for

in vitro

detection of antibodies against

Toxoplasma gondii

in porcine serum, plasma and meat juice

Catalog Number 7610230

Pub. No. MAN0013858 Rev. C.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

Toxoplasma gondii is a protozoan parasite of the family of Sarcocystiidae. Infections by the protozoan parasite are widespread in humans and many other species of

warm-blooded animals. It occurs worldwide, however, the prevalence within the animal and human populations are different between countries and holding

conditions. T. gondii undergoes an asexual reproductive cycle in all species, whereas in Felidae, the definitive host, the parasite simultaneously undergoes a sexual

cycle or replication. Carnivores and omnivores, including humans, can become infected when they eat raw or undercooked meat containing tissue cysts, tachyzoites

or consume food or water contaminated with oocysts. The cat plays an important role in the epidemiology of toxoplasmosis. However, the prevalence found in

humans cannot be maintained by the cat only, suggesting human infection through ingestion of contaminated food such as pig meat.

The Applied Biosystems™ PrioCHECK™ Porcine Toxoplasma Ab Kit is a reliable and fast diagnostic test for detection of antibodies against Toxoplasma in porcine

serum, plasma, and meat juice samples and can be used for monitoring and surveillance purposes.

Test principle

The diagnostic assay for the detection of

antibodies directed against Toxoplasma gondii in

porcine serum, plasma and meat juice samples is

based on ELISA technology. Cell culture-derived

tachyzoite antigen is coated on the ELISA plate.

Serum, plasma or meat juice samples are

incubated on the plate. A peroxidase (POD)

labeled anti-pig antibody is used for the detection

of antibodies bound to the Toxoplasma antigen.

Color development using TMB substrate

measured optically at a wavelength of 450 nm

shows the presence of antibodies directed against

Toxoplasma gondii.

The PrioCHECK™ Porcine Toxoplasma Ab Kit

follows a four step protocol, consisting of Sample

Preparation, Sample Incubation, Conjugate

Incubation and Detection. One plate with

90 prepared samples can be analyzed within

150 minutes.

Kit components

5 plate kit for 450 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, where appropriate.

Component

Description

1: Test Plate

Strip Plate. Five Test Plates are delivered in vacuum bags containing a desiccant bag.

2: Sample Diluent

Ready-to-use. Two bottles containing 60 mL of Sample Diluent. The Sample Diluent is

used to dilute the samples. Color of solution: yellow

3: Washing Fluid (10x)

10x concentrate, dilute before use. Four bottles containing 60 mL Washing Fluid (10x).

Prepare Washing Fluid working solution by mixing 1 part Washing Fluid (10x) with

9 parts demineralized water or water of equal quality.

Remark: If the Washing Fluid (10x) shows a precipitate, warm the bottle in a 30°C water

bath until the precipitate is completely redissolved. Stability of Wash Fluid working

solution: 2 weeks at 22±3°C.

4: Conjugate Diluent

Ready-to-use. One bottle contains 60 mL Conjugate Diluent. The Conjugate Diluent is

used to dilute the Conjugate. Color of solution: red

5: Conjugate (30x)

30x concentrate, dilute before use. One vial containing 2 mL Conjugate (30x). Prepare

Conjugate working solution by mixing 1 part of Conjugate (30x) with 29 parts of Conjugate

Diluent. Dilute the amount of Conjugate necessary to run the test, just prior to use.

6: Chromogen (TMB)

Substrate

Ready-to-use. One bottle containing 60 mL Chromogen (TMB) Substrate. The

Chromogen (TMB) Substrate is the substrate for the color reaction.

7: Stop Solution

Ready-to-use. One bottle containing 60 mL Stop Solution. The Stop Solution is used to

stop the color development.

8: Demineralized Water

One vial containing 10 mL Demineralized Water. Demineralized Water is used to

reconstitute the lyophilized control samples.

9: Positive Control

Lyophilized. One vial containing the lyophilized Positive Control. The Positive Control is

reconstituted by adding 200 µL of Demineralized Water (Component 9). Mix by vortexing

thoroughly and inverting the vial several times. Store at −20 to −80°C after

reconstitution. Can be aliquoted or may be freeze-thawed up to 5 times.

10: Weak Positive Control

Lyophilized. One vial containing lyophilized Weak Positive Control. The Weak Positive

Control is reconstituted by adding 200 µL of Demineralized Water (Component 9). Mix by

vortexing thoroughly and inverting the vial several times. Store at −20 to −80°C after

reconstitution. Can be aliquoted or may be freeze-thawed up to 5 times.

Note: Use of this control is optional.

11: Negative Control

Lyophilized. One vial containing lyophilized Negative Control. The Negative Control is

reconstituted by adding 200 µL of Demineralized Water (Component 9). Mix by vortexing

thoroughly and inverting the vial several times. Store at −20 to −80°C after

reconstitution. Can be aliquoted or may be freeze-thawed up to 5 times.

Additional kit contents

Package Insert

Additional material required

Use

Description(1)

General

Laboratory equipment according to national safety regulations.

• Demineralized water or water of equal

quality must be used

• Dummy plate, used for sample dilution

(e.g. clear colorless round bottom 96 well

plates) or equivalent; non-binding

• Single channel pipette (10−100 µL)

• Single channel pipette (20−200 µL)

• Multichannel pipette (5−50 µL)

• Multichannel pipette (50−300 µL)

• Pipette tips (as recommended by

pipette manufacturer)

• Solution reservoirs

• Vortex

Sample preparation

• Dedicated blood collection tubes

• Dedicated meat juice collection tubes

Analysis of results

Plate Reader, e.g. Tecan Sunrise or equivalent. The reader has to have an appropriate

filter set to read the plates at 450 nm. (Reference 620 nm).

Optional

Plate washer.

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

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

1 March 2022

Test procedure

Precautions

• National guidelines for working with animal samples must be strictly

followed.

• The PrioCHECK™ Porcine Toxoplasma Ab 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™ Porcine Toxoplasma Ab Kit,

the following aspects must be considered:

• The Test Procedure protocol must be strictly followed.

• 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 water or equal must be used for the test.

Sample preparation

• Serum and plasma can be obtained using standard methods.

• If meat juice is tested, a piece of muscle tissue e.g. 10 g (preferable tongue,

diaphragm or masseter) is either freeze-thawed in a dedicated device or

alternatively the piece of meat can be squeezed to obtain meat juice.

Sample dilution

Preparatory steps

• Reconstitute the control samples by adding 200 µL of Demineralized Water

(Component 8). Mix by vortexing thoroughly and inverting the vial several

times or use already reconstituted control samples that have been stored at

−20°C to −80°C.

Sample dilution for serum and plasma samples

1. Use a Dummy Plate or equivalent for first sample dilution (see Table 1).

2. Add 10 µL of Positive Control to wells A1 and B1 of the Dummy Plate.

3. Add 10 µL of Negative Control to wells C1 and D1 of the Dummy Plate.

4. (Optional) Add 10 µL of Weak Positive Control to wells E1 and F1 of the

Dummy Plate.

5. Add 10 µL of serum or plasma samples to the remaining wells of the Dummy

Plate.

6. Add 90 µL of Sample Diluent to each well of the Dummy Plate and mix by

pipetting up and down 5 times.

7. Add 80 µL of Sample Diluent to each well of the Test Plate.

8. Transfer 20 µL of the diluted samples and controls from the Dummy Plate to the

corresponding wells of the Test Plate and mix by pipetting up and down

5 times.

Sample dilution for meat juice samples

1. Use a Dummy Plate or equivalent for first sample dilution (see Table 1).

2. Add 10 µL of Positive Control to wells A1 and B1 of the Dummy Plate.

3. Add 10 µL of Negative Control to wells C1 and D1 of the Dummy Plate.

4. (Optional) Add 10 µL of Weak Positive Control to wells E1 and F1 of the

Dummy Plate.

5. Add 90 µL of Sample Diluent to each control in the Dummy Plate.

6. Add 20 µL of each meat juice sample to the remaining wells of the Dummy Plate.

7. Add 180 µL of Sample Diluent to each meat juice sample in the Dummy

Plate.

8. Add 80 µL of Sample Diluent to the control wells of the Test Plate (see Table 1).

9. Transfer 20 µL of the diluted control samples from the Dummy Plate to the

corresponding wells of the Test Plate.

10. Transfer 100 µL of the diluted meat juice samples from the Dummy Plate to

the corresponding wells of the Test Plate.

Sample incubation

1. Incubate the samples on the Test Plate for 60±1 minutes at room temperature

(22±3°C).

2. Wash the Test Plate four times with 300 µL 1x Washing Fluid working solution.

Remark: If you use a plate washer be sure that no needles are clogged.

Conjugate incubation

Preparatory steps

• Dilute the needed amount of Conjugate (30x) 30 fold in Conjugate Diluent (e.g add

400 µL Conjugate to 11.6 mL Conjugate Diluent for one full plate).

Conjugate incubation

1. Add 100 µL of the diluted Conjugate to each well of the Test Plate.

2. Incubate the Test Plate for 60±1 minutes at 22±3°C.

3. Wash the Test Plate four times with 300 µL 1x Wash Fluid working solution.

4. Remove remaining liquid by clapping out the plate on a paper towel.

Remark: Remaining wash solution might disturb the substrate reaction in the

detection steps!

Detection

Substrate reaction

1. Add 100 µL of the Chromogen (TMB) Substrate to each well of the Test Plate.

2. Incubate the Test Plate for 15±1 minutes at 22±3°C.

3. Add 100 µL of the Stop Solution to each well of the Test Plate.

Remark: Add the Stop Solution in the same order and time as the Chromogen

(TMB) Substrate solution was dispensed.

Note: The color of the Positive Controls will change from blue to yellow.

Detection

1. Shake the Test Plate shortly (5−10 s) either on an orbital shaker (~300 rpm) or

manually on the working bench.

2. Read the Test Plate in the ELISA reader at 450 nm within 15 minutes.

Recommendation: Use a reference filter at 620 nm.

Result interpretation

Calculation of results

PP = OD450 Sample - OD450 NC

OD450 PC - OD450 NC × 100

Validation criteria

• The mean OD450 of the Positive Controls must be >1.2.

• The mean OD450 of the Negative Controls must be <0.3.

• If the Weak Positive Control was used, the mean percentage of positivity (PP)

of the Weak Positive Controls must be >35%.

If these criteria are not met, the results are invalid and the plate has to be retested.

Interpretation of results

• Results obtained above or equal to the cut-off of 20 PP are considered positive.

• Results obtained below the cut-off of 20 PP are negative.

Recommended plate layouts

The following plate layouts allow for efficient transfer of pre-diluted samples

and controls from the Dummy Plate to the Test Plate (P — Positive Control;

N — Negative Control; W — Weak Positive Control (optional); S — Sample).

Table 1 Plate layouts: Dummy Plate and Test Plate

W or S

Customer and technical support

Technical support: visit thermofisher.com/askaquestion

Visit thermofisher.com/support for the latest in services and support.

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

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 of Pub. No. MAN0013858 (English)

Rev.

Date

Description

C.0

1 March 2022

The mean OD

450

of the Negative Controls was updated.

B.0 27 August

2020

• Updated the protocol to make the use of the Weak Positive Control

optional.

• Added a section for recommended plate layouts.

A.0

September

2019

• New document. Converted the legacy document

(Toxoplasma_ELISA_PI_v1.3_e_131107.doc) to the current document

template, with associated updates to the publication number, limited

license information, warranty, trademarks, and logos.

• The product name was changed from PrioCHECK® Toxoplasma Ab

porcine to PrioCHECK

™

Porcine Toxoplasma Ab Kit.

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 Porcine Toxoplasma Ab Kit User guide

Brand: Thermo Fisher Scientific

Model: PrioCHECK Porcine Toxoplasma Ab Kit

Type: User guide

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Thermo Fisher Scientific PrioCHECK Porcine Toxoplasma Ab Kit User guide

Thermo Fisher Scientific PrioCHECK Porcine Toxoplasma Ab Kit User guide

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