Thermo Fisher Scientific PrioCHECK Besnoitia Ab 2.0 serum plasma bovine 7610530 User guide

Brand: Thermo Fisher Scientific

Model: PrioCHECK Besnoitia Ab 2.0 serum plasma bovine 7610530

Type: User guide

PrioCHECK® Besnoitia Ab 2.0

ELISA for in vitro detection of antibodies against Besnoitia besnoiti in bovine serum and plasma samples

5 plate kit for 450 samples

©Prionics AG

Version 2.1_e

For in vitro veterinary diagnostic use only

Store at 5±3°C

Product No.: 7610530

Introduction

Bovine besnoitiosis is caused by infection with Besnoitia

besnoiti, an apicomplexan protozoan parasite. The trans-

mission of the disease and the life cycle of this parasite are

to date not fully understood. There might be a mechanical

transfer of the disease by biting flies, such as Tabanus and

Stomoxys. All cattle breeds, independent of sex and age,

can be infected. Affected cattle undergo different stages of

disease with a range of symptoms including skin thicken-

ing, swelling, hair loss and skin necrosis. Infection in bulls

can lead to infertility. In severe cases the disease leads to

the animal’s death. Cysts of Besnoitia besnoiti (200 -600

μm in diameter) are found in the conjunctival sclera of the

eyes, subcutaneous tissue, fascia and mucosa of the

infected animals and remain in the animal for years.

Besnoitia besnoiti infections are likely to cause substantial

economic losses and bovine health impairment. Therefore

the identification of infected animals and of subclinical

infected animals is crucial.

The PrioCHECK® Besnoitia Ab 2.0 has been designed to

detect antibodies specific for Besnoitia besnoiti in serum

and plasma of bovines. The PrioCHECK® Besnoitia Ab 2.0

is an excellent tool to screen cattle herds in order to

identify infected animals. Identifying infected animals is

important to avoid spreading of the disease and also to

avoid introducing infected animals into a new herd.

Test Principle

The diagnostic assay for the detection of antibodies

directed against Besnoitia besnoiti in bovine serum and

plasma is based on ELISA technology. Besnoitia antigen is

coated on the ELISA plate. Serum or plasma samples are

incubated on the plate. A peroxidase (POD) labeled anti-

bovine antibody is used for the detection of antibodies

bound to the antigen. Color development using TMB

substrate measured optically at a wavelength of 450 nm

shows the presence of antibodies directed against Bes-

noitia besnoiti. The PrioCHECK® Besnoitia Ab 2.0 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 120 minutes.

Kit Components

Store kit at 5±3°C until expiry date. See kit label for actual

expiry date. The shelf life of diluted, opened or reconsti-

tuted components is noted below, where appropriate.

Chemical hazard data are available in section “Safety

Regulations and R&S Statements” (Appendix IV).

Component 1

Test Plate (Strip Plate)

Five Test Plates are delivered in vacuum bags con-

taining a desiccant bag.

Component 2

Sample Diluent (ready-to-use)

Two bottles containing 60 ml of Sample Diluent.

Color of solution: yellow.

Component 3

Washing Fluid (20x)

(20x concentrated, dilute before use).

Two bottles containing 60 ml Washing Fluid. Prepara-

tion of Washing Fluid working solution see Appendix II.

Stability of Washing Fluid working solution: 2 weeks at

22±3°C.

Component 4

Conjugate Diluent (ready-to-use)

One bottle containing 60 ml Conjugate Diluent.

Color of solution: red.

Component 5

Conjugate (30x)

(30x concentrated, dilute before use).

One vial containing 2 ml of Conjugate. Preparation of

Conjugate working solution see Appendix II.

Dilute just before use.

Component 6

Chromogen (TMB) Substrate (ready-to-use)

One bottle containing 60 ml of Chromogen (TMB)

substrate.

Component 7

Stop Solution (ready-to-use)

One bottle containing 60 ml Stop Solution.

Component 8

Positive Control (ready-to-use)

One vial containing 350 µl.

Component 9

Weak Positive Control (ready-to-use)

One vial containing 350 µl.

Component 10

Negative Control (ready-to-use)

One vial containing 350 µl.

Additional Kit Contents:

Package Insert

Additional Material Required

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 plate) or equivalent

 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:

 Appropriate blood collection tubes for serum or

plasma collection.

Analysis of Results:

 Plate Reader, e.g. Multiscan EX or equivalent.

The reader has to have an appropriate filter set to

read the plates at 450 nm.

Optional:

 Plate washer, e.g. Tecan HydroFlex™, 96

Plate Washer™ or equivalent.

Test Procedure

Precautions

National guidelines for working with animal samples must

be strictly followed. The PrioCHECK® Besnoitia Ab 2.0

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.

Chemical hazard data are available in section “Safety

Regulations and R&S Statements” (Appendix IV).

Notes

To achieve optimal results with the PrioCHECK® Besnoitia

Ab 2.0, 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 water or equal must be used for the test.

SOLUTION PREPARATION

Washing Fluid working solution

Dilute Washing Fluid (20x) (Component 3) 1/20 in

demineralized water or water of equal quality. Mix well

until a clear solution is obtained. See Appendix II for

dilution table.

Remark: If the Washing Fluid (20x) shows precipitates,

warm the bottle up in a 30°C water bath until the

precipitates are completely redissolved.

Conjugate

Dilute the needed amount of Conjugate (30x) (Com-

ponent 5) 1/30 in Conjugate Diluent (Component 4).

See Appendix II for dilution table.

Remark: The Conjugate working solution must be

prepared just before use.

SAMPLE PREPARATION

1.1 Use a Dummy Plate or equivalent as described

under the point “Additional Materials”.

1.2 Add 20 µl of Positive Control (Component 8) to

wells A1 and B1 of the Dummy Plate.

1.3 Add 20 µl of Weak Positive Control (Compo-

nent 9) to wells C1 and D1 of the Dummy Plate.

1.4 Add 20 µl of Negative Control (Component 10)

to wells E1 and F1 of the Dummy Plate.

1.5 Add 10 μl of serum or plasma samples

to the remaining wells of the Dummy

Plate.

Package Insert

PrioCHECK

Besnoitia Ab 2.0

1.6 Add 90 μl of Sample Diluent (Component 2) to

each well of the Dummy Plate except for the

A1 to F1 and mix thoroughly by microtiter

shaker for 1 minute or by pipetting up and

down at least 5 times.

1.7 Add 90 μl of Sample Diluent (Component 2) to

each well of the Test Plate (Component 1).

1.8 Transfer 10 μl of controls and the diluted

samples from the Dummy Plate to the corre-

sponding wells of Test Plate (Component 1).

Mix thoroughly by microtiter shaker for 1 min-

ute or by pipetting up and down at least 5

times.

SAMPLE INCUBATION

2.1 Incubate the samples on the Test Plate for

60±1 minutes at room temperature (22±3°C).

2.2 Wash the Test Plate 3 times with 300 μl Wash-

ing Fluid working solution (see Appendix II).

Remark: If you use a plate washer, be sure that no

needles are clogged.

CONJUGATE INCUBATION

3.1 Add 100 μl of the diluted Conjugate to each

well of the Test Plate.

3.2 Incubate the Test Plate for 30±1 minutes at

22±3°C.

3.3 Wash the Test Plate 3 times with 300 μl Wash

Fluid working solution (see Appendix II).

3.4 Remove remaining fluid by placing the plate

face down on clean filter paper and by beating

it several times on the absorbent paper.

Remark: Remaining wash solution might disturb the

substrate reaction in the detection steps!

DETECTION

Incubation with Chromogen (TMB) Substrate

4.1 Add 100 μl of the Chromogen (TMB) Substrate

(Component 6) to each well of the Test Plate.

4.2 Incubate the Test Plate for 15±1 minutes at

22±3°C.

4.3 Add 100 μl of the Stop Solution (Component 7)

to each well of the Test Plate.

Remark: Start the addition of stop solution 15±1

minutes after the first well was filled with Chromogen

(TMB) Substrate solution. Add the Stop Solution in the

same order as the Chromogen (TMB) Substrate

solution was dispensed.

READING OF THE TEST AND CALCULATING THE

RESULTS

5.1 Shake the Test Plate briefly (5 -10 s) either on

an orbital shaker (~300 rpm) or manually on

the working bench.

5.2 Measure the optical density (OD) of the wells in

the plate reader at 450 nm within 15 minutes

after color development has been stopped.

5.3 Calculate the mean OD450 value of the Controls

(Wells A1 and B1 = mean of Positive Controls,

wells C1 and D1 = mean of Weak Positive

Controls, wells E1 and F1 = mean of Negative

Controls).

Recommendation: Use a reference filter at 620 nm.

RESULT INTERPRETATION

Calculation of results

ODPC = mean value of Positive Control

ODNC = mean value of Negative Control

The percent positivity (%P, PP) of PC is considered as

100%.

Validation criteria

6.1 The mean OD450 of the Positive Controls must

be > 1.0.

6.2 The mean percentage of positivity (PP) of the

Weak Positive Controls must be > 35.

6.3 The mean OD450 of the Negative Controls must

be < 0.15.

If these criteria are not met, the results are invalid and

the samples have to be retested.

Interpretation of results

PP ≥ 23 (Positive)

Results obtained above or equal the cut-off of 23 PP are

positive.

17 ≤ PP < 23 (doubtful)

Results obtained above or equal 17 PP until below 23 PP are

doubtful.

We recommend testing a new sample from this doubtful animal

4 weeks later.

PP < 17 (Negative)

Results obtained below the cut-off of 17 PP are negative

Appendix I

Notice

This manual is believed to be complete and accurate at the time

of publication. In no event shall Prionics AG be liable for

incidental or consequential damage in connection with or

arising from the use of this manual.

Liability

Prionics AG warrants its products will meet their applicable

published specification when used in accordance with their

applicable instructions and within the declared products life

time. Prionics AG makes no other warranty, expressed or

implied. There is no warranty of merchantability or fitness for a

particular purpose. The warranty provided herein and the data,

specifications and descriptions of Prionics AG products appear-

ing in Prionics AG published catalogues and product literature

may not be altered except by express written agreement signed

by an officer of Prionics AG. Representation, oral or written,

which are inconsistent with this warranty or such publications

are not authorized and if given, should not be relied upon.

In the event of a breach of the foregoing warranty, Prionics

AG’s sole obligation shall be to repair or replace, at its option,

the applicable product or part thereof, provided the customer

notifies Prionics AG promptly of any such breach. If after

exercising reasonable efforts, Prionics AG is unable to repair or

replace the product or part, then Prionics AG shall refund to the

customer all monies paid for such applicable product or part.

Prionics AG shall not be liable for consequential, incidental,

special or any other indirect damages resulting from economic

loss or property damage sustained by any customer from the

use of its products.

Prionics AG and Prionics Lelystad B.V. are ISO 9001:2008

certified.

Appendix II

Preparation of Washing Fluid working solution and

Conjugate Solution

Washing Fluid working solution

Mix indicated volumes of demineralized water and

Washing Fluid (20x) (Component 3).

 Transfer 50 ml Washing fluid (20x) (Component

3) to a 2 l bottle.

 Add 950 ml of demineralized water and mix.

Remark: If the Washing Fluid (20x) shows precipita-

tions, warm the bottle up in a warm water bath (ap-

proximately 30°C) until all salts are completely redis-

solved.

Conjugate working solution

Mix indicated volumes of Conjugate (30x) (Component

5) with the appropriate amount of Conjugate Diluent

(Component 4) to obtain the desired amount of Conju-

gate.

Appendix III

Pipetting Schemes

Recommended pipetting scheme for Dummy Plate

and Test Plate.

Appendix IV

Safety Regulations and R&S Statements

National Safety Regulations must be strictly followed.

R&S Statements

Component 1

Test Plate

Hazard Code: This product is not classified according to EU

regulations.

Component 2

Sample Diluent (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 3

Washing Fluid (20x)

This product does not have to be labelled due to the calculation

procedure of the “General Classification guideline for prepara-

tions of the EU” in the latest valid version.

Component 4

Conjugate Diluent (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 5

Conjugate (30x)

Hazard Code: This product is not classified according to EU

regulations.

Component 6

Chromogen (TMB) Substrate (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 7

Stop Solution (ready-to-use)

Hazard Code: R35: Causes severe burns.

S26: In case of contact with eyes, rinse immediately with plenty

of water and seek medical advice.

S36/37/39: Wear suitable protective clothing, gloves and

eye/face protection.

S45: In case of accident or if you feel unwell, seek medical

advice immediately (show the label where possible).

Component 8

Positive Control (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 9

Weak Positive Control (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 10

Negative Control (ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Contact

Prionics AG

Wagistrasse 27a

CH-8952 Schlieren-Zurich

Switzerland

Tel. +41 44 200 20 00

Fax +41 44 200 20 10

www.prionics.com

[email protected]

Prionics Lelystad B.V.

P.O. Box 2271

8203 AG Lelystad

The Netherlands

Tel: +31 320 714 000

Fax: +31 320 714 029

For our distribution network, please refer to

www.prionics.com

No. of

Plates ml Conjugate needed ml of Conjugate (30x)

ml of Conjugate

Diluent

1 12 ml = 0.4 ml + 11.6 ml

2 24 ml = 0.8 ml + 23.2 ml

3 36 ml = 1.2 ml + 34.8 ml

4 48 ml = 1.6 ml + 46.4 ml

5 60 ml = 2.0 ml + 58.0 ml

123456789101112

Positive

Control

Sample

Positive

Control

Sample

Weak

Positive

Control

Sample

Weak

Positive

Control

Sample

Negative

Control

Sample

Negative

Control

Sample

Thermo Fisher Scientific PrioCHECK Besnoitia Ab 2.0 serum plasma bovine 7610530 User guide

Thermo Fisher Scientific PrioCHECK Besnoitia Ab 2.0 serum plasma bovine 7610530 User guide

Thermo Fisher Scientific PrioCHECK Besnoitia Ab 2.0 serum plasma bovine 7610530 User guide

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