Standard Operating Procedure for virus neutralisation test

EURL-PPR SOP for VNT PPR 1

Standard Operating Procedure for

virus neutralisation test - PPR

This standard operating procedure describes how to perform Virus Neutralisation Test (VNT) to

identify the presence of specific antibodies against PPR virus into serum samples.

I. Method principle

Detection of serum specific antibodies against PPR virus by in vitro neutralisation of the viral

cytopathic effect on cell culture.

II. Abbreviations

VNT : Virus Neutralisation test

PPR : Peste des Petits Ruminants

TCID50 : 50% Tissue Culture Infective Dose

FCS: Foetal Calf Serum

CPE: Cytopathic Effect

III. Materials

a) Equipment

- Pipettes: P100, P200, P1000, multichannel 50-300 µl and graduated pipettes - Sterile tubes - 96 well sterile microplates for tissue culture - Troughs for reagent distribution - Water bath set at 56°C - Incubator 37 °C with 5% CO2 atmosphere - Laminar flow cabinet - Inverted microscope - Refrigerator and freezer - Vortex - Centrifuge


b) Reagents - VERO Cells - Titrated PPR virus suspension - Cell culture medium (MEM) (for example: MEM. ref : 21090055, Invitrogen, Fischer Scientific, France) - L Glutamine (for example: L Glutamine. ref : 25030032 Invitrogen, Fischer Scientific, France - Foetal Calf Serum (for example Dutscher ref : Pan, 3302-P280901 ) - Antibiotic-antimycotics (for example: Antibiotic-antimycotic ref: 15240062 Invitrogen, Fischer Scientific, France) - Test sera - Cleansinald à 0,5% - Alcohol 60% - Disinfectant (for example Ecodiol) - Positive control serum diluted 1:10 - Negative control serum diluted 1:10

IV. Procedure

a) Working sheet

Take a record sheet and fill it in according to the samples to be tested. All samples must be

recorded individually (see plate)

The first page is labelled with date, virus and operator. Note which PPR virus stock is used, the

virus titre and the virus dilution (inverse log) and all information required at this stage.

b) Preparation of the medium used in the plate

Prepare a sufficient volume of medium: MEM + 2% antibiotic/antimycotic + 1% L Glutamine

without serum.

c) Preparation of the serum to be tested and the control serum

In case the sera must be filtrated use a 1 ml syringe and a 13 mm Eppendorf filter with 0,22

µm or 0,45 µm porosity.

o Decomplement sera by heath at 56 °C for 30 min in a water bath.

o Dilute the serum samples (test and control) 1/10 in medium (without serum) in Eppendorf

tubes


d) Dilution of the virus

o Remove the viral suspension from -80°C just before use.

o The virus stock, with a known titre, should be diluted to give a 103 TCID50 /ml. Use MEM

without serum but with 2% antibiotics antimycotics and 1% glutamine. 100 l (10 2 TCID50) of

this viral dilution will be added to each well, therefore prepare enough volume according to the

number of plate you want to do (you need 10 ml for one plate)

o For the control plate, take five sterile tubes labelled (1,2,3,4,5) and put 1.5 ml of the

1000TCID50/ml virus dilution in the tube (1) and add 1350 µl of MEM without serum in the other four

tubes (2,3,4,5) and prepare serial dilutions (1:10) of the virus at 10 3 TCID50/ml (doing a serial

dilution of the 100 TCID50 virus suspension ( 150 µl in 1350 µl of medium, thus giving tubes

containing:

o 1.350 ml of virus 100 TCID50



o 1.5 ml of virus 0,1 TCID50

o 1.650 ml of virus 0,01 TCID50

e) Preparation of the Vero cells suspension

o Vero cells are trypsinized. After counting the viable cells, they are diluted to make a

suspension of 4x105 cell/ml in MEM + 10% CSF + 2% antibiotic/antimycotic + 1% L Glutamine

(You need 5 ml for one plate)

f) Addition in the test plate

Take a 96 well tissue culture plate. One plate allows testing 6 sera tested in duplicate in serial

dilutions (1:2)

o Addition of medium: 100 µl of medium without serum is distributed in all wells except in line

B (second line)


Scheme for medium distribution

Serum I Serum II Serum III Serum IV Serum V Serum VI

1 2 3 4 5 6 7 8 9 10 11 12

Serum

control A

100

µl/well

100

µl/well

100

µl/well

100

µl/well

100

µl/well

100

µl/well

Serial

Dilution

1/10 B - - - - - - - - - - - -

1/20 C

100

µl/well

100

µl/well

100

µl/well

100

µl/well

100

µl/well

100

µl/well

1/40 D

1/80 E

1/160 F

1/320 G

1/640 H

Addition of samples:

o 100 µl of dilution 1/10 of each serum to be tested in the first line of the plate (line A), each

sample is added in duplicate (A1, A2 …), the same sample in A1 and A2, the second sample in A3

and A4 …. A5 and A6…..A7 and A8. Change pipette tips for every sample. This line will not receive

the virus.

o 200 µl of dilution 1/10 of each sample to be tested in the second line of the plate (line B),

each sample is added in duplicate (B1, B2 …), the same sample in B1 and B2, the second sample

in B3 and B4 …. B5 and B6…..B7 and B8. Change pipette tips for every sample.

o With multichannel pipette set at 100 µl perform serum dilution by removing 100 µl from line

B to line C and ….from line D to E and from F to G … H. Discard from the last row. Thus the first

dilution of the sera is 1/10 and the last dilution is 1/640.


Scheme for samples distribution

Serum I Serum II Serum III Serum IV Serum V Serum VI

1 2 3 4 5 6 7 8 9 10 11 12

Control

serum A

100 µl au

1/10

Serial

Dilution

1/10 B 200 µl

1/20 C

1/40 D

1/80 E

1/160 F

1/320 G

1/640 H

o Add 100 µl of virus (100 TCID50) in each well except in the line A.

o Incubate in the incubator (37°C, 5% CO2).

o After 1 hour add in each well of the plate 50 µl of Vero cells suspension of (4x105 cell/ml).

o Incubate 7 days at 37 °C and 5% CO2.

g) Addition in the control plate

For each test a control plate has to be done.

The control plate allows the verification of the quality of the cells (control cell), viral dose

employed (virus dilution), its specificity (negative control), sensitivity (positive control) and

detection limit (limit dilution of the positive control).

100 µl

100µl

100 µl

100 µl

100 µl

100 µl

100 µl


Medium distribution:

- 100 µl of medium without serum from line A to line E ( ten well per line : column 2 to 11),

- 200 µl in line F,

- 100 µl in lines G and H, in the column 2 to 6 and 8 to 12.

-

Scheme for medium distribution in the control plate:

1 2 3 4 5 6 7 8 9 10 11 12

Virus

Dilution

100 TCID50 A

100 µl/well

10 TCID50 B

1 TCID50 C

0,1 TCID50 D

0,01 TCID50 E

Cell control F 200 µl/well

Positive control G - 100 µl/well

- 100 µl/well

Negative control H - -

1/10 1/20 1/40 1/80 1/160 1/320 1/10 1/20 1/40 1/80 1/160 1/320

Control’s dilution Control’s dilution

Distribution of control sera:

- line G : well 1 and well 7 add 200 µl of the positive control diluted (1/10)

- line H : well 1 et well 7 add 200 µl of the negative control diluted (1/10)

- line G et H prepare a serial dilution (1:2) of the sera: transfer 100 µl from well 1 to well 2 and so on up to well 6. Do the same from well 7 to well 12. Discard the last 100 µl from wells G6, G12, H6 and H12.


Scheme for control sera distribution in the control plate:

1 2 3 4 5 6 7 8 9 10 11 12

Virus

Dilution

100 TCID50 A - - - - - - - - - -

10 TCID50 B - - - - - - - - - -

1 TCID50 C - - - - - - - - - -

0,1 TCID50 D - - - - - - - - - -

0,01 TCID50 E - - - - - - - - - -

Cell control F - - - - - - - - - -

Positive control G 200

µl

200

µl

Negative control H 200

µl

200µl

1/10 1/20 1/40 1/80 1/160 1/320 1/10 1/20 1/40 1/80 1/160 1/320


Virus distribution:

From the different viral dilutions prepared ((d) dilution of the virus) add to:

- lines A, G and H : 100 µl of virus 100 TCID50,

- line B : 100 µl of virus 10 TCID50,

- line C : 100 µl of virus 1 TCID50,

- line D : 100 µl of virus 0,1 TCID50,

- line E : 100 µl of virus 0,01 TCID50.

100 µl 100µl 100 µl 100 µl 100 µl

100 µl 100 µl 100 µl 100 µl 100 µl

100µl

100 µl 100 µl 100 µl 100µl 100 µl

100 µl 100 µl 100 µl 100 µl 100 µl

100µl


Scheme for the distribution of the virus in the control plate:

1 2 3 4 5 6 7 8 9 10 11 12

Virus

Dilution

100 TCID50 A 100 µl/wells

10 TCID50 B 100 µl/wells

1 TCID50 C 100 µl/wells

0,1 TCID50 D 100 µl/wells

0,01 TCID50 E 100 µl/wells

Cell control F - - - - - - - - - -

Positive control G 100 µl/wells 100 µl/wells

Negative control H

1/10 1/20 1/40 1/80 1/160 1/320 1/10 1/20 1/40 1/80 1/160 1/320


o Incubate in the incubator (37°C, 5% CO2).

o After 1 hour add in each well of the plate 50 µl of the suspension of Vero cells (4x105 cell/ml).

o Incubate 7 days at 37 °C and 5% CO2.

h) Observation of the cytopathic effect

After 7 days incubation period, observe each well with the inverted microscope and note on the

working sheet the cytopathic effect with a sign “ +”

Confirm the reading after 10 days

If CPE is not observed then a sign ” –“ is used

The results are registered on the computer.


V - Results

a) Test acceptance criteria

No sign of bacterial (yellow colour) or fungal (patch of mould) contamination should be observed

on the plate. The colour of the medium must be orange /pink. If it is yellow or purple it means a a

defect in the CO2 supply of the incubator.

The assay is acceptable if for the control plate:

o The real viral dose is above or equal 20 TCID50 /100 µl and lower or equal to 316 TCID50 /100 µl. The

real viral dose equal the inverse of log of N:

5,0dilutionper or wellsnumber

wellspositive ofnumber

N

- 100% of CPE for the line A (virus 100 TCID50), - at least 80% of CPE for the line B (virus 10 TCID50), - between 0 et 100% CPE for the line C (virus 1 TCID50), - maximum 50% CPE for the line D (virus 0. 1 TCID50), - 0% CPE for the line E (virus 0.01 TCID50), - 0% CPE for the line F (cell control) without virus, - 100% CPE for the line H (negative control) (virus 100 TCID50),

In addition, no viral dilution should have more CPE than any lower viral dilution.

b) Calculation of the serum sample titre

Calculation is done automatically in the work sheet.

On the two dilutions series made for a serum sample, the titre can only be calculated if the difference

between the two negative results (no CPE) and the two positive results (CPE) is not more than two dilutions:

the different acceptable possibilities are shown in the table hereunder. In all other cases, the titre is not

calculated and the sample is declared inconclusive.


Example of acceptable cases:

Dilutions Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Test 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2

1/10 - - - - - - - - - - - -

1/20 - - - - - - + - - + - -

1/40 + + - + + - - - + - + +

1/80 + + + + + - + + + + - -

1/160 + + + + + + + + + + + +

The titre of the sample is equal to the inverse of the first dilution where the two wells are positives plus

inverse of the last dilution where the two wells are negative, all being divided by two.

2

negatives wellsith twodilution wlast

1

positives wellsith twodilution wfirst

1

T

Therefore, in the above example, the titre of sample 1 and 2 are respectively 30 and 50.

If the progression of CPE is not regular in the two series of dilution, (jump of well as in the case of samples 3

to 6 where CPE’s do not match with dilutions), the calculation of the titre is done as before, after having

moved the positivity of the well to the dilution just above (higher dilution) . Therefore, the titre of sera 3, 4,

5 and 6 are respectively 60, 50, 30 and 60.

Example of moving positive wells:

Dilutions Sample 3 Sample 4 Sample 5 Sample 6

Test 1 Test 2 Test 1 Test 2 Test 1 Test 2 Test 1 Test 2

1/10 - - - - - - - -

1/20 - - + - - + - -

1/40 + - - - + - + +

1/80 + - + + + + - -

1/160 + + + + + + + +


3- Calculation of uncertainty

Uncertainty is calculated automatically in the work sheet.

The uncertainty is equal to the titre calculated as described above, less the inverse of the last dilution where

the two wells are negatives (before moving any positive result)

wellsnegatives ith twodilution wlast

1I T

Example for calculation of titre and uncertainty:

Sample A Sample B Sample C

Sample control - - - - - -

1/10 - - - - - -

1/20 + - - - - -

1/40 + + + + - +

1/80 + + + + - +

1/160 + + + + + +

1/320 + + + + + +

1/640 + + + + + +

Titre

± uncertainty

(40 + 10)/2 = 25

±

(25 - 10) = 15

(40 + 20)/2 = 30

±

(30 - 20) = 10

(80 + 40)/2 =60

±

(60 - 20) = 40


4- Sample result interpretation

- A serum is declared positive if its titre is equal or above 10.

- If the initial dilution of a sample is above 1/10 because the volume is not sufficient and if the titre of

this sample is seen not to go beyond this dilution, the titre is given with the sign ‘‘≤’’ and it is declared

inconclusive.

- For samples with a titre go beyond the dilution scale, the titre is given with the sign ‘‘≥’’ and it is

declared positive.

- The value of the uncertainty is taken into consideration for the expression of the final qualitative

result.

Interpretation is done as described below.

Titre Uncertainty Interpretation

7.5 ± 2.5 negative

12.5 ± 7.5 Positive near to the cut-off

15 ± 5 Positive near to the cut-off



30 ± 10 Positive

30 ± 20 Positive

Documents

Standard Operating Procedure for virus neutralisation test