Civtest Avis Ibd

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CIVTESTTM SUIS ADV (gE)

Test kit for the detection of antibodies to glycoprotein gE of Pseudorabies Virus, in serum samples.

Code / Microplates / Assays
0121 / 5 / 460

CIVTEST™ SUIS ADV (gE)

Test kit for the detection of antibodies to glycoprotein gE of Pseudorabies Virus, in serum samples.

General information

In control program of the Aujeszky disease it is of great interest to make a differenciation between vaccinated pigs from those infected with the field strain of PRV, as both infected and vaccinated are seropositive.

Most vaccinal strains used in vaccination program are well known attenuated strains with a deletion of the gene sequence coding for the glycoprotein gE.

Consequently, vaccinated pigs with such vaccine lack antibodies against the gE antigen.

The differentiation is thus based on the presence of antibodies to gE in infected pigs but the absence of these antibodies in vaccinated pigs.

gE deleted vaccinated pigs, non infected with field strain of PRV, doesn’t have any antibodies against gE protein. So this population of animals will give a negative result with the ELISA CIVTESTTM SUIS ADV (gE).

gE deleted vaccinated pigs, infected with field strain of PRV, do have antibodies against gE protein. So this population of animals will give a positive result with the ELISA CIVTESTTM SUIS ADV (gE).

The specificity of the CIVTESTTM SUIS ADV (gE)has been evaluated on the following deleted vaccine :

AUSKIPRA – BK (Hipra Laboratory, S.A.)

AUSKIPRA – GN (Hipra Laboratory, S.A.)

GESKALONE gE (Merial)

SUVAXYN (Solvay)

AUSKIMMUNE gE (SKF – Beecham)

INGELVAC (Boehringer Ingelheim)

NOBI-VAC AUJESZKY gE (Intervet)

NOBI PORVAC (Intervet)

Test principle

CIVTESTTM SUIS ADV (gE) is a blocking ELISA test.

The plates are coated with PRV antigen. This antigen is derived from an inactivated strain, known to posses the glycoprotein gE. The indicator used for the detection of presence or absence of antibodies against gE is a monoclonal antibody (mAb) specifically directed against gE. The mAbs are conjugated with the HRP enzyme.

If antibodies are absent in the serum sample, the gE antigen coated on the plate will remain free, and the conjugated mAbs subsequently added will bind. The enzyme present will catalyze the added substrate, which results in a color reaction. The intensity of the reaction is measured at 450 nm.

If antibodies against gE are present in the serum, these antibodies will bind to the gE antigen in the well. This will block the possibility of the conjugated mAbs subsequently added to react with the antigen. The unbound conjugated mAbs will be removed by the washing procedure followed, resulting in no color reaction when the substrate is added.

So the test will discriminate between animals infected (score positive, no coloration) and animals vaccinated with a gE deleted vaccine (score negative, yellow coloration).

- Reagent 1: Plate coated with inactivated PRV antigen.

- Reagent 2: Negative Control PRV gE.

- Reagent 3: Positive Control PRV gE.

- Reagent 4: PRV gE HRP Conjugate. Ready to use, pink coloration.

- Reagent A: PBS-Tween concentrate (10x).

- Reagent C: Substrate Solution. Ready to use.

- Reagent D: Stop Solution. Ready to use.

Procedure

1. All reagents should equilibrate to room temperature 18-25°C before use. Let the reagents stand in room temperature at least one hour prior to use.

2. SAMPLES AND CONTROLS DISTRIBUTION

- Add 100µl of the positive control and 100µl of the negative control into each of the appropriate wells, respectively. It is recommended to run a duplicate for each reference. For example: positive control in well A1 and B1 negative control in well C1 and D1.

- Add 100µl of undiluted serum sample to each of the appropriate wells.

3. SAMPLES INCUBATION

Seal the microtitre plate and incubate for 1 hour at 37±2ºC.

4. WASHING

Wash the plate 3 times. (see section washing procedure).

5. CONJUGATE DISTRIBUTION

Add 100 µl per well of the anti PRV gE conjugate. Incubate for 30 minutes at 37±2ºC.

6. WASHING

Wash the plate 3 times. (see section washing procedure).

7. SUBSTRATE DISTRIBUTION

Add 100µl of substrate solution to each well and incubate for 15 minutes at 18-25°C, in darkness.

8. STOP DISTRIBUTION

Add 100µl of stop solution to each well. Add the stop solution in the same order as the substrate solution was added in step 7.

9. O.D. MEASURE

Within 15 minutes after adding the stopping solution read the plate at 450nm on a microplate photometer.

Calculation

Calculate the mean OD of Negative Control : ODm NC

Calculate the inhibition % of test samples and Positive Control according to the following formula.

(O.D.m NC – O.D. sample)

INH % = ------x 100

O.D. m NC

Validation

To ensure validity of the test :

1.- The difference between O.D.m control negative and O.D.m control positive should be higher than 0,6.

O.D.m negative control – O.D.m positive control > 0,6

2.- The positive control should have a INH. % over 60.

% INH. PC > 60%

Interpretation of the results

INTERPRETATION / % INH.
POSITIVE / % INH. > 45
NEGATIVE / % INH. < 40
DOUBTFUL / 40 £ % INH £ 45

Graphic presentation of results

Dossier
Kit CIVTEST SUIS ADV (gE)
N° of sample / 10
INH % / 74 %
CV (%) / 33 %
Identification / INH % / Class / Interpretation
1 / Neg
2
3
4
5
6
7
8
9
10
Minimum
Maximum
Cut off
Class limits Table
Classes / Parameter / Size (n)) / Size (%)
0
1
2
3
4
5
6
7
8
9

Materials needed but not provided

1. Precision pipettes (range from 5 to 1000 µl).

2. Disposable pipette tips.

3. Distilled water.

4. Wash bottle or automatic platewasher.

5. Microplate reader with a 450 nm filter.

6. Centrifuge (2000 g)

7. Centrifuge tubes and microtubes.

8. Vortex or equivalent.

Precautions

1. Carefully read and follow all instructions.

2. Store the kit and all reagents at +2°C to +8°C.

3. All reagents should equilibrate to 18°C to 25°C before use.

4. Unused microtitre wells should be stored sealed within the enclosed plastic bag at +2°C to +8°C.

5. Handle all materials as though capable of transmitting infectious disease.

6. Do not intermix components or instruction booklets from different test kits.

7. Care should be taken to prevent contamination of kit components.

8. Do not use test kit beyond expiration date.

9. Do not eat drink or smoke where specimens or kit reagents are being handled.

10. Use a separate pipette tip for each sample.

11. Do not pipet by mouth.

12. Include positive and negative reference on each plate or test strip series.

13. Use only distilled water for preparation of reagent.

14. The stop solution contains H2SO4 which is a strong acid and can cause burns. Handle with care.

15. Burn all unused biological materials before disposal.

16. For in vitro diagnostic use only.

Sample preparation

100 µl of pur serum is required for each sample well. Fresh, refrigerated or previously frozen serum may be tested. For confirmation purposes, it is suggested that each sample should be run in duplicate.

Preparation of reagents

- Microtitre wells, anti PRV gE conjugate, positive control serum , negative control serum, substrate solution, stop solution are ready to use.

- PBS-Tween Concentrate (10x) : the PBS-Tween solution is diluted 1/10 in distilled water (or higher quality water). The diluted solution can be stored at +4°C up to 3 days, or one month frozen at –20°C.

Example : For 250 ml ready to use solution, dilute 25ml 10x concentrate PBS-Tween, with 225 ml distilled water and mix well.

Washing procedure

1. All washing will be conducted with a plate washer or a multichannel pipette which allows the distribution up to 300µl. Diluted PBS-Tween solution will be used for the washing (see section preparation of reagent).

2. If a manual washing is performed, proceed as follow :

- empty the contents of the plate by inversion.

- distribute 300µl per well of PBS-Tween x1 solution.

- shake the plate thoroughly.