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1. Introduction
There are a number of methods of influenza virus titration based
on its biological characteristics, such as the virus ability to agglutinate red blood cells (RBCs) of different species (1). The ability of
infectious and noninfectious virus particles to agglutinate RBC is
the basis of the hemagglutination assay (HA). The plaque assay,
50% tissue culture infectious dose (TCID50), and 50% egg infectious
dose (EID50) are methods used to determine the amount of infectious
Yoshihiro Kawaoka and Gabriele Neumann (eds.), Influenza Virus: Methods and Protocols,
Methods in Molecular Biology, vol. 865, DOI 10.1007/978-1-61779-621-0_3, Springer Science+Business Media, LLC 2012
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2. Materials*
2.1. Hemagglutination
Titration Assay
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The materials needed for the TCID are the same as those listed for
the microneutralization assay.
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3. Methods
3.1. Hemagglutination
Assay
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A B C D E F G H
2-fold
serial
dilution
100ul virus
50ul
Fig. 1. The 96-well microtiter plate layout for the hemagglutination titration assay.
Table 1
Recommended time of incubation at 2025C
for the hemagglutination assay with the use
of different species of RBCs
Avian
Mammalian
Chicken
Turkey
Guinea pig
Human
type O
Concentration
0.5%
0.5%
0.75% v. 75%
0.75%
Microtiter plate
Incubation time
30 min
30 min
1h
1h
Appearance of
control cells
Buttona
Buttona
Halo
Halo
When tilted at a 45 angle the RBCs in the V-well plate will flow
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Wells
4 5
HA
Titer
1
2
4
8
16
32
64
128
256
512
1024
2048
Results of HA
Well # HAU
1
128
2
64
3
64
4
32
5
128
6
256
7
256
8
0
Table 2
Example of EID50 calculation using the ReedMuench method
Log of virus
dilution
Infected
samples
Cumulative
positive (A)
Cumulative
negative (B)
Ratio of A/(A + B)
Percent
infected (%)
3/3
6/6
100
2/3
3/4
75
1/3
1/4
25
0/3
0/6
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3. Thaw virus in cool water. Perform tenfold dilution series starting at 101 and diluting virus samples down to 1010 (or less)
dilution in plaque assay wash medium. Changing tips between
dilutions is required. Inoculate 6-well tissue culture wells in
duplicate with 100 ml diluted virus samples and gently rock tissue culture plate to cover monolayer with inoculums. Incubate
plates at 3337C for 3060 min (see Note 11).
4. After incubation, wash wells twice with room temperature (20
25C) plaque assay wash medium, taking care not to add medium
directly onto the monolayer as this may disrupt the cells.
5. Add 1 ml of 2 mg/ml TPCKtrypsin working stock to 2
plaque assay medium. Mix (1:1) 2 plaque assay medium with
Lonza SeaKem Le Agarose and immediately add 2 ml of this
mixture to each inoculated well. Allow to solidify at room
temperature (2025C). Incubate 6-well tissue culture plates
at 3337C. Observe MDCK plates daily with an inverted
microscope for plaque formation.
6. After 72 h, remove the agar plug from each well using sterile
forceps. Pipette 2 ml of 70% ethanol into each well and incubate 20 min at room temperature (2025C) to fix the MDCK
cell monolayer. Remove ethanol and add 1 ml crystal violet
solution to each well. Incubate at room temperature (2025C)
for 10 min to stain MDCK cell monolayer. Remove crystal violet solution and wash wells with water to rinse away excess stain
solution. Allow plates to dry overnight at room temperature
(2025C) prior to counting plaques.
7. Count plaques (Fig. 3) in each well and determine the PFU per
milliliter using the following formula: PFU/ml = number of
plaques dilution factor 10 (see Note 12). The PFU/ml calculation should be based on the dilution of virus sample that gives
>10 plaques per well but is still countable. The number of plaques
are counted, and then multiplied by the reciprocal of the amount
Fig. 3. Well of a 6-well plate indicating plaques visualized. The plaques will appear as clear
circular areas on the background of the crystal violet-stained monolayer.
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1. The serum samples should be treated with RDE (see Note 13)
to remove nonspecific inhibitors of hemagglutination. Add 3
volumes of RDE to the tube with 1 volume of serum. Incubate
in 37C water bath for 1820 h. Remove tubes from the 37C
bath and place them in a 56C water bath for 30 min to inactivate the RDE (see Note 14).
2. Remove tubes from 56C water bath and allow to equilibrate
to room temperature (2025C). Add 6 volumes of physiological saline (0.85% NaCl).
3. To remove nonspecific agglutinins from the serum samples,
add 1 volume of packed RBCs to 20 volumes of RDE-treated
serum. Mix thoroughly and incubate at 48C for 1 h shaking
tubes periodically to resuspend cells. Centrifuge at 400 g for
10 min at 48C.
3.4.2. Standardization
of Antigens
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Table 3
To adjust antigens based on the results of the back titration.
If the back-titration result is less that 8 HA units, add more
antigen and if greater than 8 HA units add buffer
Interpretation
of HA units
7 volumes
5 volumes
3 volumes
2 volumes
volume
No adjustment
volume
16
Equal volume
16
2 volumes
32
3 volumes
32
5 volumes
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Fig. 4. Interpretation of an HAI assay. Columns 1, 2, 3, and 7 have HAI titers of 1:10.
Column number 4 has an HAI titer of 80. Column 5 has an HAI titer of 20 and column 6 has
an HAI titer of 40. Column 8 was the negative control.
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Table 4
Dilutions of cell suspensions for passage in T-162-cm2 flasks
Dilution
When confluent
4.0
56 106
~24 h (1 day)
2.0
1.752 106
~48 h (2 days)
1.0
57 105
~72 h (3 days)
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virus dilution at the cut-off point. We typically use 200 times the
virus dilution at the cut-off point for avian influenza viruses such as
H5N1 to obtain 100 TCID in 50 ml and 400 times the virus dilution for seasonal H3N2 and seasonal or pandemic H1N1 viruses to
obtain 200 TCID in 50 ml (see Note 18). The minimum dilution
of the virus stock for the microneutralization assay is 1:100. The
virus titration is done on day 1 and the ELISA to detect viralinfected cells is done on day 2.
1. Rapidly thaw a vial of virus at 37C and immediately place on
ice. Virus should be thawed just before use for optimal infectivity.
Test each virus at two or more different starting dilutions.
Dilutions of 102, 103, and 104 in virus diluent are suggested
depending upon how well the virus replicates in eggs or cells.
Test each dilution on separate microtiter plates.
2. Add 100 ml of virus diluent to all wells, except column 1, of a
96-well microtiter plate. Add 146 ml of the virus starting
dilution to all wells in column 1. Transfer 46 ml serially from
column 1 through column 11. Change pipette tips between
wells. After mixing, discard 46 ml from column 11. This dilution
scheme results in log10 dilutions. Dilutions will be 102, 102.5,
103, 107 if the starting dilution was 102. Column 12 contains virus diluent only (no virus) and is the cell control (CC).
3. Stack plates and cover with an empty plate. Place in 37C, 5%
CO2 incubator for 1 h to mimic the virus + serum incubation
step of the microneutralization assay.
4. Prepare MDCK cells for use in assay as described. Add 100 ml of
diluted cells to each well of the microtiter plate. Each well will
contain 1.5 104 cells. Incubate at 37C in 5% CO2 for 1820 h.
5. The procedures for day 2 of the TCID determination, plate
fixation, 1 antibody, 2 antibody, and substrate, are the same
as these procedures for the microneutralization assay.
6. To analyze the data, calculate the median absorbance (O.D.)
of the cell controls (column 12) and multiply by two to obtain
the cut-off value. Prepare a table like Table 5. Once all test
wells have been scored positive or negative for virus growth
based on the cut-off value, the TCID of the virus suspension
can be calculated by the method of Reed and Muench (4).
Record the number of positive and negative values at each
dilution. Select the region of the data where the transition
from all wells positive at one dilution to all wells negative
occurs. This region is shown in the box in Table 5. Calculate
the cumulative numbers of positive wells at each dilution.
The cumulative number positive is obtained by adding the
positives at each dilution starting at the bottom. Calculate
the cumulative numbers of negative wells at each dilution.
The cumulative number negative is obtained by adding the
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Table 5
Calculation of TCID by the ReedMuench method
Observed value (O.D.)
Cumulative value
# Positive
# Negative
S Positive
2.5
103
103.5
Dilution
10
10
S Negative
Ratio
% Positive
100
16
16/16
4.5
10
8/9
89
105
1/9
11
105.5
16
0/16
10
106.5
107
10
89 50
0.5 = 0.5 0.5 = 0.25
89 11
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Fig. 5. Overview of microneutralization assay. The main steps of the microneutralization assay are shown. Steps 1 and 2,
the neutralization portion of the assay and step 3, the infection of MDCK cells with non-neutralized virus, are done on day
1. Steps 4 and 5, the detection of virus infected cells using an ELISA, are done on day 2.
3.7.1. Microneutralization
Assay Procedure
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Fig. 6. Image of developed microneutralization assay plate. Ten serum samples were
tested on this plate in columns 1 though 10 as noted at the bottom of the plate. The serum
dilution is shown on the right-hand side of the microtiter plate. Column 11 shows the virus
back titration (BT). The virus control (VC) is shown in the first four wells of column 12; the
cell control (CC) in the second four wells of column 12. The titers obtained for these serum
samples after data analysis are shown across the top of the plate.
14. The data are analyzed as follows: The VC and CC medians are
determined for each plate. In order for the ODs on each plate
to be considered valid, the CC must have a median OD of 0.2.
The VC must have a mean OD of 0.8. The virus back titration
is evaluated to determine if the working dilution of virus used
in the assay contained the desired amount of virus. The cut-off
value for the virus back titration is the mean of the VC median
and the CC median. This is the same cut-off that is used to
evaluate neutralizing antibody titers. The dilution of the first
well below the cut-off value is the back titration titer. The dilutions in the back titration wells are beginning at well A: 1:2,
1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. In general, back
titration titers of 16, 32, and 64 are acceptable (see Note 21).
15. Neutralizing antibody titers are determined by calculating the
cut-off value to determine a 50% neutralizing antibody titer for
each plate based on the equation: (median O.D. of VC + median
O.D. of CC)/2 = X, where X = the 50% cut-off value. All values
below or equal to X are positive for neutralization. Read each
column which contained diluted serum from the bottom,
beginning at well H. Note the first well with an OD of less than
the 50% cut-off. The reciprocal serum dilution corresponding to
that well is the 50% neutralization antibody titer for that serum
sample. Serum dilutions are well A 1:10, well B 1:20, well C
1:40, well D 1:80, well E 1:160, well F 1:320, well G 1:640, and
well H 1:1,280. Serum samples with a titer of >1,280 may be
repeated beginning at a 1:80 starting dilution to obtain an
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Neutralization by negative
control sera
Weak or no neutralization
by positive control sera
Solution
Check the antibodies and substrate
Prepare fresh buffers
Redetermine virus TCID or adjust the dilution of virus used or repeat
and do not forget to add virus to VC wells
Check incubator temperature and CO2 level
Thaw a new vial of cells, do not allow cells to enter stationary phase
Possible cause(s)
Problem
Table 6
Potential problems associated with interpretation of the microneutralization assay
3
Influenza Virus Titration, Antigenic Characterization, and Serological Methods
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4. Notes
1. Blood from a farm should be prescreened, it is recommended
to find a commercial source. The RBCs used for all testing
should be as fresh as possible. The blood should be preserved
in Alsevers or sodium citrate at a 1:1 dilution.
2. In the 1990s, human influenza A (H3N2) lost its ability to
agglutinate chicken RBCs (13) RBCs such as turkey or guinea
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Acknowledgments
The microneutralization protocol presented here is based on the
work of Thomas Rowe and others who developed the original
microneutralization protocol for avian influenza H5N1 viruses.
Thanks is expressed to Yaohui Bai and Li Cronin for their work
determining the number of MDCK cells needed to obtain flasks of
cells at the appropriate confluency for the microneutralization assay.
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Quantitative aspects of the red blood cell agglutination test for influenza virus. J. Exp. Med.
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3. Miller, G.L. (1965) Improved measurement of
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4. Reed, L.J. and H. Muench. (1938) A simple
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5. Tobita, K., A. Sugiura, C. Enomoto, and M.
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