Thermo Fisher Scientific PrioCON conjates Classical Swine Fever User guide

Brand: Thermo Fisher Scientific

Model: PrioCON conjates Classical Swine Fever

Type: User guide

PrioCON conjugates

Diagnostic procedures for Classical Swine fever

Detection and differentiation of pestiviruses in pigs

©Prionics AG

Version 1.1_e

For in-vitro veterinary diagnostic use only

Product numbers: 7610010, 7610015, 7610020,

7610025, 7610030, 7610035

Introduction

To diagnose classical swine fever (CSF) solely on

clinical symptoms and post mortem lesions is not

always possible. Significant differences exist between

symptoms caused by classical swine fever virus

(CSFV) strains of high, intermediate and low virulence.

Therefore, diagnosis of CSF must be made in the

laboratory where CSFV can be detected in tissue

specimens, isolated in an appropriate cell system, and

where sera can be tested for antibodies against CSFV.

The immuno-fluorescence test (IFT) is used to detect

CSF-antigen in cryostat sections of organ specimens.

This test is performed with a conjugate prepared from

a polyclonal anti-CSF serum, therefor it cannot differ-

entiate between pestiviruses. To differentiate pestiviru-

ses, monoclonal antibodies (MAbs) are used in an

immunoperoxidase test (IPT). Serology is performed

with the –PrioCHECK® CSFV Ab ELISA for the

detection of CSFV antibodies (Ab). The neutralization

peroxidase- linked assay (NPLA) can be used to

confirm the positive results obtained in the Pri-

oCHECK® CSFV Ab.

Described are the laboratory techniques and procedu-

res used to detect and differentiate pestiviruses, and to

detect and differentiate antibodies directed against

pestiviruses, according to the national Dutch reference

laboratory for Classical Swine Fever Virus (CIDC, the

Netherlands).

Test Principles

THE DIRECT IMMUNOFLUORESCENCE TEST (IFT)

USING THE PRIOCON FITC PAN-PESTIVIRUS

The IFT detects viral antigens. The test is performed

on cryostat tissue sections of the tonsil, spleen, kid-

ney, and the distal part of the ileum. Tissue sections

are incubated with a fluorescein labeled anti-pestivirus

globulin (conjugate). The sections are examined for

fluorescent cells with an ultraviolet light microscope.

Since the IFT employs a conjugate prepared from a

polyclonal anti-CSFV globulin it cannot differentiate

between antigens of various pestiviruses. Therefore, a

positive result with the IFT must be followed by an

immunoperoxidase test (IPT) using monoclonal anti-

bodies (MAbs) that can differentiate CSFV from other

pestiviruses.

THE IMMUNOPEROXIDASE TEST (IPT) USING

PRIOCON HRPO PAN-PESTIVIRUS, PRIOCON

HRPO 21.2, PRIOCON HRPO 44.3 AND PRIOCON

HRPO 63.19

To detect whether a positive result with the IFT is

caused by CSF field virus, CSF vaccine virus, bovine

viral diarrhoea virus (BVDV) or border disease virus

(BDV), differentiation with monoclonal antibodies

(MAbs) specific for CSF and the Chinese vaccine

strain of CSF is performed.

Cryostat sections of the tonsil are separately incubated

with 3 different peroxidase labeled MAbs (coded

PrioCON HRPO 21.2, PrioCON HRPO 44.3, Pri-

oCON HRPO 63.19 and a peroxidase labeled polyclo-

nal antibody PrioCON HRPO Pan-pestivirus.

It is recommended to incubate all conjugates on

positive reference sections as well. Subsequently all

sections are stained with chromogen/substrate (AEC,

see Annex).

PrioCON HRPO 21.2 recognizes all CSFV strains.

PrioCON HRPO 44.3 does not recognize CSF vaccine

virus. Both conjugates do not recognize BVDV and

BDV. If the reaction with PrioCON HRPO 21.2, and

PrioCON HRPO 44.3 is negative and the sections

incubated with the PrioCON HRPO Pan-pestivirus

are positive, the diagnosis is BVDV or BDV. PrioCON

HRPO 63.19 detects only the Chinese strain of CSFV

and is added to the test when the vaccine virus of

CSFV is suspected.

THE NEUTRALIZATION PEROXIDASE LINKED

ASSAY (NPLA) USING PRIOCON HRPO 39.5

The NPLA is a neutralization test for the detection of

antibodies against CSFV or BVDV and is performed in

microtiter plates. Sera are inactivated and two-fold

dilutions (in duplicate) are incubated with the appro-

priate virus strain. After incubation, CSFV or BVDV

susceptible cells are added to the serum-virus mixture.

The cells grow to confluency in the wells of a micro-

titer plate. Virus, which is not neutralized by antibod-

ies, will propagate in the cells. CSFV propagates in

PK-15 cells and BVDV in MDBK (or EBTr) cells. The

dilution of the virus for use in the NPLA is critical since

it determines the sensitivity of the test. Appropriate

references must be incorporated in the test to estab-

lish sensitivity and specificity. Very important is the

absence of BVD virus as well as BVDV antibodies in

the foetal bovine serum (FBS) that is used in the

growth medium for the cells. After fixation, the cells

are incubated with a direct HRPO conjugate (39.5) to

detect the virus. Subsequently, the chromogen-

substate is added. The test serum is negative for

CSFV-antibodies when the cytoplasm of the cells is

stained deeply red. The test can be read macroscopi-

cally or, in case of doubt, with an inverted microscope.

Storage conditions

The undiluted FITC conjugates should be stored at

–20oC upon arrival.

The undiluted HRPO conjugates should be stored at

4oC upon arrival.

Test Procedures

PROCEDURE OF THE DIRECT IMMUNOFLUORES-

CENCE TEST (IFT)

1.1 Cut sections (4 µm) of the organ specimens on a

cryostat microtome. The sections are caught on

a coverslip.

1.2 Include a CSFV positive- and negative section in

the test to verify conjugate activity and back-

ground staining.

1.3 Dry the sections 15 minutes at 37°C or fixate the

sections 10 minutes in acetone.

1.4 Rinse the sections once in phosphate buffered

saline (PBS).

1.5 Prepare working dilution of conjugate in PBS.

Place the cover slips in a humid chamber and

dispense the working dilution of the PrioCON

FITC Pan-pesti in PBS on the sections.

1.6 Incubate 30 minutes at 37°C in the humid cham-

ber.

1.7 Wash the sections 5x 2 minutes in PBS/0,05 %

Tween 80 at room temperature (20 – 25°C).

1.8 Drain the cover slips and mount them on an

object slide with Mounting Buffer (from Prionics

Lelystad B.V.) (0,5 M carbonate buffer (pH 9.5)

diluted 1:10 in 100 % glycerol).

1.9 Examine the sections for fluorescent cells

(cytoplasm) with an ultraviolet light microscope.

1.10 Interpretation of the test.

In case of a positive IFT cytoplasmatic fluorescence

can be seen in:

- The epithelial cells which outline the tonsillar crypts;

- Lymphoid cells in the peri-arterial-lymphocyte-sheath

(PALS) of the spleen;

- The epithelial cells of the ascending limb of the loop

of Henle in the kidney;

- The epithelial cells of the villi in the ileum;

- Other cells of epithelial origin.

A negative IFT does not exclude CSF. In case of a

suspicious clinical picture, tissue specimens from

additional pigs from the suspected farm must be

investigated. Simultaneously, virus isolation can be

attempted.

Annex IFT

To decrease non-specific background staining, speci-

mens can be counterstained, using Counterstain

(0,01% Evan’s Blue) from Prionics Lelystad B.V.

- Dilute Counterstain 50x in the working dilution of the

conjugate.

THE IMMUNOPEROXIDASE TEST (IPT) USING

PRIOCON HRPO PAN-PESTIVIRUS, PRIOCON

HRPO 21.2, PRIOCON HRPO 44.3 AND PRIOCON

HRPO 63.19

2.1 Cut eight more sections (4µm) of the IFT-positive

organ (usually the tonsil but kidney sections pro-

vide a good picture as well). Sections are caught

on a cover slip.

2.2 Dry de sections for 15 minutes at 37°C or fixate

the sections in acetone for 10 minutes.

2.3 Rinse the sections once in PBS/0,05 %

Tween80.

2.4 Prepare working dilution of conjugate in PBS (pH

7.6) + 0.05% Tween80 + 4% horse serum. Incu-

bate the sections (including CSFV positive- and

negative reference sections) with working dilu-

tion of PrioCON conjugates:

-2 sections with PrioCON HRPO 21.2

-2 sections with PrioCON HRPO 44.3

-2 sections with PrioCON HRPO 63.19 (if vac-

cine virus is suspected)

-2 sections with PrioCON HRPO Pan-

pestivirus.

2.5 Incubate for 1 hr at 37°C in the humid chamber.

2.6 Wash the sections 6x 10 seconds in PBS/0,05 %

Tween 80.

2.7 Rinse the sections once in 0,05 M sodium

acetate (pH 5,0).

2.8 Stain the sections with a freshly prepared chro-

mogen/substrate solution

(3-amino-9-ethyl-carbazole (AEC)) for 5 - 15

minutes at room temperature (20 – 25 °) (See

annex IPT).

2.9 Rinse the sections once in 0.05 M sodium

acetate (pH 5,0) and mount them on an object

slide.

2.10 Examine the sections with a light microscope.

A dark red stained cytoplasm of the epithelial

cells which outline the tonsillar crypts indicates

recognition of the virus by the respective conju-

gates, and is considered as a positive test result.

2.11 Interpretation of the test.

IFT

IPT Diagnosis

21.2

44.3

Pan-pesti

CSF

CSF vaccine

virus

BVDV/BDV

*Confirm virus specificity with MAb 63.19.

Package Insert

PrioCON conjugates

Annex IPT

Preparation of chromogen/substrate solution

(AEC)

Stock solution of chromogen:

1) 4 mg 3-amino-9-ethyl-carbazole (AEC)

2) 1 ml N,N-di-methyl-formamid

Prepare the following just before use:

3) 19 ml 0.05 M Na-acetate (pH 5.0)

4) Add per 20 ml AEC solution 10 ul 30 % H2O2

5) Filtrate the solution through a 5 micron acrodisc

filter (Gelman Sciences prod. no.4199).

Keep the AEC stock solution at 4°C in the dark.

PROCEDURE OF THE NPLA

Preparation of serum dilutions in microtiter plates

The starting dilution is 1:25 (OIE manual prescribes

1:10) and is made in a dummy plate. These dilutions

are transferred to the test plate in which the sera are

further diluted.

3.1 Dispense 240 µl growth medium

3.2 Dispense 10 µl of each test serum per well in the

growth medium (serum dilution is then 1:25) of

the dummy plate.

To test the sera in duplicate, dispense test se-

rum 1 in wells A1 and A2, serum 2 in wells A3

and A4 etc.

3.3 Shake the plates for 10 seconds on a microplate

shaker.

3.4 Transfer 100 µl of the 1:25 serum dilution in row

A of the dummy plate to the corresponding wells

of row A of the first test plate. Transfer 100 µl of

the 1:25 serum dilution in row B of the dummy

plate to the corresponding wells of row A of the

second test plate etc.

3.5 Dispense 50 µl of the growth medium in the rows

B through H of the test plate(s).

3.6 Make a two-fold dilution starting in row A of the

test plates by transferring 50 μl of row A to row

B, stir and transfer 50 µl to row C, etc.

3.7 Make a two-fold dilution of a strong positive con-

trol serum (in octuple) in the columns 1 through

6 of a separate (reference) plate.

3.8 Make a two-fold dilution of a weak positive

control serum in columns 7 through 11 of the

control plate (in octuple).

3.9 Dispense 100 µl of growth medium in column 12

of the reference plate. The wells in this column

serve as negative cell reference.

Incubation of serum dilutions with the (predetermi-

ned) virus working dilution

3.10 The working dilution must contain approximately

100 TCID50 per 50 µl of the virus (predetermined

by titration).

3.11 Add 50 µl of the virus working dilution to all wells

of the test plates containing the serum dilutions.

3.12 Dispense 50 µl growth medium in the wells of

columns 1 till 5 of the virus control plate.

3.13 Make a tenfold dilution (10-1 – 10-5) of the virus

working dilution in test tubes and dispense 50 µl

per dilution (in octuple) in the columns 1 till 5 of

the virus reference plate.

3.14 Incubate serum dilutions with virus working

dilution in the test plates and reference plates for

1 hr at 37°C in 5 % C02 incubator.

3.15 Add to the wells of all plates 50 µl growth medi-

um containing 2.105 cells per ml.

3.16 Incubate the cultures in a 5 % CO2 incubator at

37°C for 4 days (CSFV) or 5 days (BVDV). The

cells should grow gradually to confluency in 3

through 5 days.

Fixation and staining procedure

3.17 Discard the growth medium and rinse the plates

once in 0,15 M NaCl. Drain the plates thoroughly

by knocking them on a towel.

3.18 Fix the cells by heating the plates for 1 hr at

80°C.

3.19 Rinse the plates once in 0,15 M NaCl.

3.20 Prepare working dilution of PrioCON HRPO 39.5

in PBS + 0.05 % Tween 80 + 4 % horse serum.

Add to the wells of all plates 50 µl pre-diluted

conjugate. If a neutralization test for the detec-

tion of antibodies against BVDV is performed,

the MDBK cells can be stained with a prede-

termined dilution of a PO labeled CSFV- or

BVDV immune serum).

3.21 Incubate 1 hr (CSFV) or 2 hrs (BVDV) at 37°C.

3.22 Wash the plates 5x in PBS + 0,05 % Tween 80.

3.23 Add to all wells of the plates 50 µl of the chro-

mogen-substrate solution 3-amino-9-ethyl car-

bazole (AEC; See annex IPT).

3.24 Incubate the AEC on the cells for 15 - 30

minutes.

Reading and interpretation of the test

The test can be read macroscopically. In case of doubt

the monolayers should be examined with an inverted

microscope.

325 Deep red stained cytoplasm of the cells indicates

that the test serum is negative for antibodies.

Does the test serum contain antibodies, virus

growth is prevented by neutralization and the cy-

toplasm of the cells is not stained.

3.26 Calculate, via the titration of the virus working

dilution in the virus control plate, the number of

TCID50 according the method of Reed &

Muench. The number of TCID50 must be be-

tween 40 and 400.

3.27 Calculate the titers of the strong and weak positi-

ve control sera in the serum control plates accor-

ding to the method of Reed & Muench.

3.28 Determine the titer of the test sera. The titer of a

serum is expressed as the reciprocal of the

highest dilution that prevents virus growth in 50

% of the wells.

A serum with a titer in the CSFV-NPLA of

≥

25 is

considered positive.

A serum with a titer in the BVD-NPLA of

≥

5 is

considered positive.

Ordering information

- PrioCON FITC Pan-pestivirus

art# 7610035

- PrioCON HRPO Pan-pestivirus

art# 7610030

- PrioCON HRPO anti-CSFV (21.2)

art# 7610010

- PrioCON HRPO anti-CSFV (44.3)

art# 7610020

- PrioCON HRPO anti-CSFV C-strain (63.19)

art# 7610025

- PrioCON HRPO anti-CSFV (39.5)

art# 7610015

- Priotest Mounting Buffer

art# 7610037

- Priotest Counterstain (0.01% Evans Blue)

art# 7610036

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 partic-

ular purpose. The warranty provided herein and the

data, specifications and descriptions of Prionics AG

products appearing in Prionics AG published cata-

logues 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 rea-

sonable 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 appli-

cable 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 prod-

ucts.

Prionics AG and Prionics Lelystad B.V. are ISO

9001:2000 certified companies.

Appendix II

References

1. Terpstra, C. and Wensvoort, G. (1988a) Natural

infections of bovine viral diarrhoea virus in pigs,

associated with signs resembling swine fever.

Res. Vet. Sci., 45: 137-142.

2. Wensvoort, G. and Terpstra, C. (1988) Bovine

viral diarrhoea infections in piglets born from

sows vaccinated against swine fever with con-

taminated vaccine. Res. Vet. Sci., 45: 143 - 148.

3. Terpstra, C. (1978). Detection of C-strain virus in

pigs following vaccination against swine fever.

Tijdschr. Diergen. 103, 678 - 684.

4. Wensvoort, G., Terpstra, C., Boonstra, J.,

Bloemraad, M. and van Zaane, D. (1988). Pro-

duction of monoclonal antibodies against swine

fever virus and their use in laboratory diagnosis.

Vet. Microbiol. 12, 101 - 108.

5. Wensvoort, G., Bloemraad, M. and Terpstra, C.

(1988) An enzyme-immunoassay, employing

monoclonal antibodies and detecting specifically

antibodies against classical swine fever virus.

Vet. Microbiol. 17, 129 - 140.

6. Terpstra, C., Bloemraad, M. and Gielkens, A.L.J.

(1984). The neutralizing peroxidase-linked assay

for detection of antibody against swine fever vi-

rus. Vet. Microbiol. 9, 113 - 120.

7. Wensvoort, G., Terpstra, C., de Kluijver, E.P.,

Kragten, C., and Warnaar, J.C. (1989) Antigenic

differentiation of pestivirus strains with mono-

clonal antibodies against hog cholera virus. Vet.

Microbiol., 21: 9 - 21.

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

info@prionics.com

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Thermo Fisher Scientific PrioCON conjates Classical Swine Fever User guide

Brand: Thermo Fisher Scientific

Model: PrioCON conjates Classical Swine Fever

Type: User guide

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Thermo Fisher Scientific PrioCON conjates Classical Swine Fever User guide

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