Quantification

of Virus and
Neutralization
Test
Center for Vaccine Development

By:
Putri Aliya Ahadini
Made Retna Paramita S
 01 Medium preparation and Cell Culture

What we 02 Quantification of Virus by Plaque Assay

have 2

learned? 03 Quantification of Virus by

Real Time - PCR

04 PRNT
01
Medium 3

Preparation
 Mix it
with
Add 5 ml stirrer 2
of milliQ Mix with until 3
water stirrer hours

The
4
Steps
Add 5 Add 11 Divide into 10
packs of 1 L gram of smaller botol,
MEM Sodium be careful it
Powder Bicarbonate must not
to adjust contaminated
the pH of
the medium
RESULT 5
Vero Cell 6

Culture
 Observe Discard Rinse Add 1 ml
the cells the twice by of Trypsine
growth adding versene, Discard the
media 5 ml of gently rock Trypsine
PBS back versene

Using a pipette, Add 5ml

Gently tap Incubate at room The
the flask to temperature 7
dissociation the of MEM Steps
clumps of cells detach the around
by repulsing cells 3-10 minute
approximately
20 times

Takes 0,9 ml PBS Takes 100 μL to

and 0,1 ml cell hemocytometer
culture and mix and count under
it by vortex the microscope
 8

Counting
Cell
 Observe Discard Rinse Add 1 ml
the cells the twice by of Trypsine
growth adding versene, Discard the
media 5 ml of gently rock Trypsine
PBS back versene

Using a pipette, Add 5ml

Gently tap Incubate at room The
the flask to temperature 9
dissociation the of MEM Steps
clumps of cells detach the around
by repulsing cells 3-10 minute
approximately
20 times Takes 2 ml
of cells
and 3 ml
of MEM
Takes 0,9 ml PBS Takes 100 μL to Incubate at
and 0,1 ml cell hemocytometer 37 C and
culture and mix and count under CO2 5%
it by vortex the microscope
 10

Result
 A step when the cell infected by
Infection of Cell the virus with on purpose.
For example virus propagation and
11

virus replication.
 Take a vero culture that contain 2,5 x 107 cells / ml

12

The
Quantification
 Take the
virus stock Take 0,2 ml of
and warm it virus to the
in incubator cell culture
flask

Shake while The

13
incubate for Steps
Add 5 ml 90 minute
of MEM

Keep in
incubator for
days
 RESULT 14

Contaminated Not
Can’t be used contaminated
Can be used
Cell before get
Infected

RESULT 15

NORMAL INFECTED
CELL CELL
 A method to stock the cell and
Tissue Culture prepare the cell to be
16
monolayer, it can be use further
out Growth Cell for many method, such as
plaque assay and PRNT.
A method to stock the cell.
This method need freezing medium that contain
DMSO 10% and also 10% MEM Complete
Medium. That medium support to keep the cell
Make a
safe and cell can grow again.
The cell stock must keep in the liquid nitrogen or
Cell 17

-80oC freezer. Stock

 Add 9 ml of Take out the
10% MEM sentrifuge
Complete Sentrifuge for tube, remove
Medium in the 10 – 15 minute, the medium, Take out all of
centrifuge to remove the cell pack is the suspension
tube. DMSO 10%. left in the tube. to the flask.

The
18
Steps

Take out the Add 1 ml of cell Add 5 ml of Add 2 ml of Incubate for 3

cell stock from stock to the 10% MEM 10% MEM until 7 days,
freezer and sentrifuge Complete Complete wait until it
melt it in 37oC tube. Medium to Medium and completely
water incubator small flask suspense it. monolayer.
tube.
02
A method to harvest the virus for

Harvest Virus years. This method keep the

structure of virus and can be used
19

again event after several years.

 Take it out Adjust the pH
with add 2 drop Put on the box,
from freezer,
of Sodium label it, and
melt it in 37oC
Bicarbonate keep in the
water
until the freezer.
incubator. medium is pink.
The 20
-80oC Steps
Keep the flask that
have made while Add 20% (1 ml) Divide into
infection of cells in of Fetal Bovine small tube,
freezer for 3 hours. Serum label it!
Make sure it is not
contaminated.
Quantification
A method to determine
of virus by the titer of virus.
21

Plaque Assay
 Prepare 6
tube and put
0,9 ml of PBS

Take 100 μL
of Zika virus
and mix it
with vortex
The
0,1 ml 0,1 ml 0,1 ml 0,1 ml 0,1 ml 0,1 ml 22
Steps
Do 10 fold
dilution

1 2 3 4 5 6
Prepare 6 well -1 -2 -3
plate that
contain the
cell culture,
-4 -5 -6
then discard
the medium

The Next
23
1 2 3 4 5 6 Steps
Add 200 μL
-1 -2 -3
from diluted
tube to well,
same as the
number -4 -5 -6

Shake while
incubate it for
90 minute
 25 ml 25 ml
solution A solution B

Prepare the
Mix solution A
Overlay
& B with
Medium
stirrer

-1 -2 -3
Take the 6
plate well after
incubated in 90 The Next
minute -4 -5 -6 24
Step

Put 2 ml of -1 -2 -3
overlay
medium in per
well -4 -5 -6

Save the
6 plate well
in incubator
for 7 days
 Discard the
medium

Stain every well -1 -2 -3

with 400 μL crystal
violet, keep at room
temperature for 30
minute -4 -5 -6 The Next
25
Steps

Wash the plate

with water

Count the
plaque
 -6
-5

Titer = no. of plaque per well (average) x 1 x dilution factor

RESULT 26

Volume of inoculum
Well -5  Titer = (18+14) x 1 x 105
2 0,2
= 0,8 x 10 7

Well -6  Titer = (3+3) x 1 x 106

2 0,2
= 1,5 x 107

The Titer is (0,8x107) + (1,5 x 107) =1,14 x 107

2
03 Quantification
of Virus by 27

Real Time PCR

 3. Real time PCR (Probe
1. Viral RNA isolation based assay)

The Steps 28

2. Reverse Transcription
 RT-PCR REACTION
Component Volume (20 µl rxn)

KAPA PROBE FAST qPCR Master Mix 80 µl

(2x)

10 µM D3 primer (DEN-3 F+DEN-3C) 4.0 µl 29

10 µM D3 probe (DEN-3 probe) 1.6 µl

KAPA RT Mix (50x) 3.2 µl

Nuclease-free water 55.2 µl
 Protocol set up based
on following table
Step Temperature Duration Cycles

cDNA 42ºC 5 min

1 30
synthesis
Inactive RT 95ºC 5 min 1
Denature 95ºC 10 sec

Annealing+d 60ºC 30 sec 40

ata
acquisition
RESULTS

31
03
a method for measuring the antibodies
PRNT titer that neutralize and prevent virus from
infecting cultured cells
32
PRINCIPLE METHOD
● The basic design of the PRNT
allows for virus antibody interaction Day 1 : Preparation of vero –
to occur in a test tube or microtiter Seeded Plates
plate, and then measuring antibody
level on viral infectivity by plating
the mixture on virus-susceptible Day 4 : Virus – Serum 33
cells Neutralisation

Day 7 : Immunostaining
FACT

1. Gold Standard
Positive 2. More specific than other
serological methods for the
diagnosis of some arbovirus. 34

1. Relative complicated to do
Negative 2. Take more time (7 days)
3. Depent on the cell-type used in
the assay
 35

RESULT
Thank you! 36