Thermo Fisher Scientific PrioCHECK CSFV Ag whole blood serum plasma tissue pig 7610050 User guide

Brand: Thermo Fisher Scientific

Model: PrioCHECK CSFV Ag whole blood serum plasma tissue pig 7610050

Type: User guide

PrioCHECK® CSFV Ag

ELISA for in vitro detection of antigen against Classical Swine Fever Virus in whole blood, serum, plasma and tissue

specimens of pigs

5 plate kit for 450 samples

©Prionics AG

Version 1.0_e

For in-vitro veterinary diagnostic use only

Store at 5±3°C

Product No.: 7610050

Introduction

Early detection of CSFV infected pigs is of high impor-

tance to contain the dissemination of CSFV. Thus,

early tracing of possibly infected herds is focused on

the detection of CSFV antigen rather than CSFV

antibodies which appear from approximately 14 days

post-infection onwards.

The PrioCHECK® CSFV Ag is a valuable diagnostic

tool to increase diagnostic confidence to eventually

establish a CSFV infection. Priority is given to mini-

mize false positive test results (high specificity and

selectivity) combined with the highest possible sensi-

tivity. The PrioCHECK® CSFV Ag can be used to

examine pig herds that are suspect for CSFV infection.

In CSF endemic countries (areas) where often vacci-

nation is applied the test can be used to trace residual

foci of CSFV.

Positive test results should be confirmed by a "gold"

standard test (e.g. isolation of the virus in cell culture).

Test Principle

The PrioCHECK® CSFV Ag is based on the double-

antibody-sandwich (DAS) principle. CSFV-specific

antibodies have been coated to eight-well strips. In a

first step Sample Diluent and Test Sample (whole

blood, serum, plasma, leucocyte concentrate or tissue

extract) are dispensed to the wells and incubated.

Secondly, the plate is washed and anti-CSFV conju-

gate of high specificity is dispensed to the wells. After

an incubation period the plate is washed and bound

conjugate is visualized by a subsequent incubation

with Chromogen (TMB) Substrate. After addition of

Stop Solution the color reaction is terminated. Color

development measured optically at a wavelength of

450 nm shows the presence of antigens of classical

swine fever virus.

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

reconstituted components is noted below, when

appropriate. Chemical hazard data are available in

section “Safety Regulations and R&S Statements”

(Appendix II).

Component 1

Test Plate

Five strip Test Plates.

Component 2

Conjugate (30x)

(30x concentrated, dilute before use). One vial con-

tains 2.2 ml Conjugate.

Diluted conjugate is not stable, prepare just before

use.

Component 3

Dilution Buffer (Ready-to-use)

One vial contains 60 ml Dilution Buffer.

Component 4

Conjugate Additive (Ready-to-use)

One vial contains 12.0 ml Conjugate Additive.

Component 5

Demineralized Water

One vial contains 10 ml Demineralized Water.

Component 6

Washing Fluid (200x)

(200x concentrated, dilute before use). One vial

contains 60 ml Washing Fluid.

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

Component 7

Sample Diluent (Ready-to-use)

Three vials, each contains 10 ml Sample Diluent.

Component 8

Reference Serum 1 (lyophilized)

One vial contains 1.5 ml Reference Serum 1 (strong

positive control).

Shelf life of the reconstituted Reference Serum 1: until

expiry date at -20°C.

Component 9

Reference Serum 2 (lyophilized)

One vial contains 1.5 ml Reference Serum 2 (blank).

Shelf life of the reconstituted Reference Serum 2: until

expiry date at -20°C.

Component 10

Reference Serum 3 (lyophilized)

One vial contains 1.5 ml Reference Serum 3 (weak

positive control).

Shelf life of the reconstituted Reference Serum 3: until

expiry date at -20°C.

Component 11

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

One vial contains 60 ml Chromogen (TMB) Substrate.

Component 12

Stop Solution (Ready-to-use)

One vial contains 60 ml Stop Solution.

Additional Kit Contents:

- 10 plate sealers

- Package Insert

- Certificate of Analysis

Additional Material Required

General:

Laboratory equipment according to national safety

regulations.

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 EIA Tray Washer or

equivalent.

Test Procedure

Precautions

National guidelines for working with animal samples

must be strictly followed. The PrioCHECK® CSFV Ag

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. For decontamination Halamid (1%),

sodium hydroxide (0.4%) or glutaraldehyde (1%) can

be used (4-5 log CSFV reduction in 6 minutes).

Test samples should be kept sterile to enable subse-

quent confirmation in the virus isolation test.

Chemical hazard data are available in section “Safety

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

Notes

To achieve optimal results with the PrioCHECK®

CSFV Ag, 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 pipet-

ting 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.

SOLUTIONS TO BE MADE IN ADVANCE

References

Reconstitute1 the References (Component 8 – 10) with

1.5 ml Demineralized Water (Component 5). After the

lyophilized Reference Sera have been reconstituted

they should be aliquotted in small vials. The vials can

be stored at -20°C until expiry date. The number of

aliquots to be made depends on the number of test

runs (max. 12 independent test runs).

Conjugate dilution

Dilute Conjugate (30x) (Component 2) in Dilution

Buffer (Component 3) supplemented with 20% (v/v)

Conjugate Additive; e.g. for one plate prepare 12 ml

(add 2,4 ml Conjugate Additive to 9,2 ml Dilution

Buffer, subsequently, add 400 µl Conjugate (30x)).

Washing solution

The Washing Fluid (Component 6) must be diluted

1/200 in demineralized water and is sufficient for a

final volume of 12 liters washing solution.

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

Remark: The washing of the wells after incubation is critical. A

blank optical density (OD of reference 2) of >0.300 most likely

can be ascribed to inadequate washing of the wells. Manual

washing of the wells must be performed at least 6 times:

dispense 300 µl washing solution to each well, empty the wells

carefully by tapping the Test Plate and refill. Omit formation of

air bubbles when filling the wells. Take care that after the last

washing any residual washing solution is removed by tapping

the Test Plate on tissue paper. When using automatic washers

and blank OD values >0.300 are obtained, wash manually as

described above.

Note: see Appendix IV for sample preparation

procedure and storage

INCUBATION OF SERUM AND TEST SAMPLE

1.1 Label each strip on the Test Plate (Component

1) with a marker pen.

1.2 Dispense 50 µl Sample Diluent (Component 7)

to all wells.

1.3 Dispense 50 µl of reconstituted reference serum

1 to well A1 and B1.

1.4 Dispense 50 µl of reconstituted reference serum

2 to well C1 and D1.

1.5 Dispense 50 µl of reconstituted reference serum

3 to well E1 and F1.

1.6 Dispense 50 µl of test sample to the remaining

wells.

1.7 Seal the Test Plate with a plate sealer and shake

the Test Plate gently.

1Reconstitution of lyophilized ingredients should be performed as follows:

- Equilibrate the vial to 22±3°C.

- With the vial in an upright position, tap the vial gently against the

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

- Open the vial.

- Add the specified amount of Demineralized Water (see label on vial).

- Replace the stopper on the vial and allow the lyophilized material to

dissolve.

- Gently agitate so that any remaining dry material is dissolved.

- Allow the lyophilized material to stand for 15 minutes at 22±3°C.

- Occasionally, gently invert vial (formation of foam should be avoided).

Package Insert

PrioCHECK

CSFV Ag

1.8 Incubate 180±5 minutes at 22±3°C.

Note: When dispensing whole blood samples take

care that no residual sample remains in pipette tip.

INCUBATION WITH CONJUGATE

2.1 Wash the Test Plate 6 times with 300 µl washing

solution per well. Tap the plate firmly after the

last wash cycle.

Note: No residual blood should be left in the wells.

2.2 Dispense 100 µl of the diluted conjugate to all

wells.

2.3 Seal the Test Plate with a plate sealer.

2.4 Incubate 60±5 minutes at 22±3°C.

INCUBATION WITH CHROMOGEN (TMB) SUB-

STRATE

3.1 Wash the Test Plate 6 times with 300 µl washing

solution per well. Tap the plate firmly after the

last wash cycle.

3.2 Dispense 100 µl Chromogen (TMB) Substrate

(Component 11) to all wells.

3.3 Incubate 15 minutes at 22±3°C. Do not shake.

3.4 Add 100 µl Stop Solution (Component 12).

3.5 Mix the contents of the wells of the plate.

Note: Start the addition of Stop Solution 15 minutes

after the first well was filled with Chromogen (TMB)

Substrate. Add the Stop Solution in the same order

and the same place as the Chromogen (TMB) Sub-

strate was dispensed.

READING OF THE TEST AND CALCULATING THE

RESULTS

4.1 Measure the optical density (OD) of the wells at

450 nm within 15 minutes after color develop-

ment has been stopped.

4.2 Calculate the mean OD450 of wells C1 and D1

(Reference 2 = OD450 blank).

4.3 Calculate the corrected OD450 of Reference 1

and all samples by subtracting the OD450 blank.

4.4 The percentage positivity (PP) of Reference 3

and of the test samples are calculated according

to the formula below.

The corrected OD450 values of all samples are ex-

pressed as percentage positivity (PP) relative to the

corrected mean OD450 value of reference 1.

corrected OD450 test sample

PP = -------------------------------------------- x 100

corrected OD450 Reference 1

RESULT INTERPRETATION

Validation criteria

5.1 The OD450 of Reference 2 must be <0.350.

5.2 The corrected OD450 of Reference 1 must be at

least 0.750.

5.3 The PP of Reference 3 must be >20%.

Note: Not meeting any of these criteria is reason to

discard the results of that specific test run.

Note: If the corrected mean OD450 of Reference 1 is

below 0.750 possibly the Chromogen (TMB) Substrate

is too cold. In that case warm the solution to 22±3°C or

incubate up to 30 minutes. If the corrected mean

OD450 of Reference 1 is above 2.000 a shorter incuba-

tion period with the Chromogen (TMB) Substrate is

recommended.

Interpretation of the percent positivity

PP = <15% (negative)

No detectable antigen present in the test sample.

PP = ≥15% (positive)

CSFV antigen is present in the test sample.

The presence of CSFV in antigen-positive samples

should be confirmed by virus isolation.

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 BV are ISO 9001:2000

certified companies.

Appendix II

Safety Regulations and R&S Statements

National Safety Regulations must be strictly followed.

Component 1

Test Plate

Hazard Code: This product is not classified according to EU

regulations.

Component 2

Conjugate (30x)

Hazard Code: This product is not classified according to EU

regulations.

Component 3

Dilution Buffer (Ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 4

Conjugate Additive (Ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 5

Demineralized Water

Hazard Code: These products are not classified according to

EU regulations.

Component 6

Washing Fluid (200x)

Hazard Code: This product is not classified according to EU

regulations.

Component 7

Sample Diluent (Ready-to-use)

Hazard Code: This product is not classified according to EU

regulations.

Component 8

Reference Serum 1 (lyophilized)

Hazard Code: This product is not classified according to EU

regulations.

Component 9

Reference Serum 2 (lyophilized)

Hazard Code: This product is not classified according to EU

regulations.

Component 10

Reference Serum 3 (lyophilized)

Hazard Code: This product is not classified according to EU

regulations.

Component 11

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

Hazard Code: This product is not classified according to EU

regulations.

Component 12

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 feel unwell, seek medical advice

immediately (show label on vial).

Appendix III

References

1) Ressang, A.A., (1973). Zbl. Vet. Med. B 20, 256 – 271.

2) Wensvoort et al, (1986). Vet. Microbiol. 12, 101 – 108.

3) Kaden et al, (1999). Berl. Münch. Tierärtzl. Wschr., 112, 52 -

57.

4) Martin et al, (1992). Prev. Vet. Med. 14, 33 – 34.

Appendix IV

Sample preparation procedure and storage

Several procedures can be followed to prepare a

tissue extract for subsequent testing in the Pri-

oCHECK® CSFV Ag and virus isolation in cell culture.

The procedure described below is adopted from the

method routinely used at the Reference Laboratory for

CSF of the Institute for Animal Health and Science in

the Netherlands.

Blood and plasma samples

Anticoagulant whole blood samples (preferably hepa-

rin blood) or plasma should preferably not be frozen

but stored at 5±3°C (max. 7 days) before testing.

Serum

Serum samples can be tested directly or after storage

at -70°C. After testing, the serum samples can be

stored at -70°C (to preserve viable virus).

Preparation of tissue extract

 Cut 1-2 grams of the tissue in small pieces and

grind these into a homogenous paste using a mor-

tar and pestle and a small amount of medium. Use

sterile sand as an abrasive when necessary.

 Per gram tissue add 9 ml Earle’s MEM (supple-

mented with 5 times the normal concentration of

antibiotics streptomycin, penicillin and mycostatin).

 Incubate 1 hour at 22±3°C.

 Centrifuge the suspension for 20±1 minutes at

6000x g.

 Carefully collect the supernatant and discard the

sediment.

 Aliquot the suspension in at least 2 vials.

 Store the vials at -70°C.

Preparation of leukocyte concentrate

Prior to testing, the collected leucocyte fractions of a

blood sample is preferably frozen (-20ºC) and thawed.

 Dispense 8 ml 0.83% NH4Cl solution in a 15 ml

tube.

 Add 4 ml anticoagulated whole blood sample.

 Mix the content of the tube and incubate 30 minutes

at 22±3°C in order to obtain complete lysis of the

red blood cells.

 Centrifuge for 10 minutes at 500x g at 22±3°C.

 Discard the supernatant.

 Re-suspend the leucocyte pellet in 400 µl Earle’s

MEM (supplemented with 5% foetal calf serum and

antibiotics streptomycin, penicillin and mycostatin).

 Store at -70°C (to preserve viable virus).

Contact

Prionics Lelystad B.V.

Platinastraat 33

P.O. Box 2271

NL-8203 AG Lelystad

The Netherlands

Tel. +31 320 714000

Fax +31 320 714029

Prionics AG

Wagistrasse 27a

CH-8952 Schlieren-Zurich

Switzerland

Tel. +41 44 200 2000

Fax +41 44 200 2010

www.prionics.com

[email protected]

For our distribution network, please refer to www.prionics.com

Thermo Fisher Scientific PrioCHECK CSFV Ag whole blood serum plasma tissue pig 7610050 User guide

Thermo Fisher Scientific PrioCHECK CSFV Ag whole blood serum plasma tissue pig 7610050 User guide

Thermo Fisher Scientific PrioCHECK CSFV Ag whole blood serum plasma tissue pig 7610050 User guide

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