Materials and Reagents

Materials
1. 96 well microtiter plates (Corning, catalog number: 3595 )
2. Tips for multichannel pipette (Gilson, model: D10 , D200 and D1000 )
3. Centrifuge tubes (SARSTEDT AG & Co, catalog number: 72.690 )
Reagents
1. Viral antigen
  Note: Viruses were amplified in embryonated eggs or Madin–Darby canine kidney (MDCK) cells. Refer to Manual for the laboratory diagnosis and virological surveillance of influenza, WHO.
  1. H1N1
    A/Ohio/83
    A/Solomon Islands/2006
    A/Ohio/07/2009
    A/Texas/05/2009-RG15
    A/Texas/18/2009-RG18
    A/California/04/2009
  2. H2N2
    A/Ann Arbor/6/60 ca
  3. H5N1
    A/Vietnam/1203/04 (VNH5N1-PR8/CDC-RG)
    A/Anhui/01/2005(H5N1)-PR-IBCDC-RG6
  4. H9N2
    A/ck/HK/G9/97(H9N2)/PR8-IBCDC-2
    A/Green-winged teal/209/TX/2009
  5. H3N2
    A/Hong Kong/68
    A/Philippines/2/1982
    A/Beijing/353/89-X109-H3N2 PR8 reassortant
    A/Beijing/32/92-R-H3N2 PR8 reassortant
    A/Johannesburg/33/94 R-H3N2 PR8 reassortant
    A/Nanchang/933/95
    A/Sydney/5/97
    A/Panama/2007/99
    A/Wyoming/3/03.rg
    A/Brisbane/10/07
  6. H7N2
    A/turkey/Virginia/2002(H7N2)/ PR8-IBCDC-5
  7. H7N9
    A/Anhui/1/2013
    A/Shanghai/2/2013
2. Cells
  Madin-Darby canine kidney (MDCK) cells (ATCC, catalog number: CCL-34 )
3. Neutralization antibody
  Human monoclonal antibody, CT149 (Celltrion INC., South Korea) from convalescent patients infected with A(H1N1)pdm09
4. Media
  1. DMEM (Invitrogen, catalog number: 11965-092 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 11965-092”.
  2. Fetal bovine serum (FBS) (VWR International, Hyclone™, catalog number: SH30070.03 )
  3. Penicillin-streptomycin (Invitrogen, catalog number: 15140-122 )
    Note: Currently, it is (Thermo Fisher Scientific, Gibco™, catalog number: 15140-122)”.
  4. L-glutamine, 200 mM solution (Invitrogen, catalog number: 25030-081 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco™, catalog number: 25030-081”.
  5. Bovine serum albumin (BSA) (fraction V, protease free) (Roche Diagnostics, catalog number: 0 3117332001 )
  6. HEPES, 1 M Buffer Solution (Invitrogen, catalog number: 15630-080 )
    Note: Currently, it is (Thermo Fisher Scientific, Gibco™, catalog number: 15630-080)”.
5. Other reagents
  a.Antibody for ELISA
  1. Primary antibodies [Anti-nucleoprotein (NP) antibodies], Mouse Anti-Influenza A antibody (Merck Millipore Corporation, catalog number: MAB8257 ) and Anti-Influenza A antibody (Merck Millipore Corporation, catalog number: MAB8258 )
  2. Secondary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Kirkegaard & Perry Laboratories, Inc., catalog number: 074-1802 )
  b.Phosphate buffered saline (PBS) (Gibco, catalog number: 14190 )
  c.Tween-20 (Merck Millipore Corporation, catalog number: 8.17072 )
  d.Antibody diluent (Teknova, catalog number: D5120 )
  e.Acetone (Sigma-Aldrich, catalog number: 270725 )
  f.3, 3’, 5, 5’-Tetramethylbenzidine (TMB) (Sigma-Aldrich, catalog number: T0440 )
  g.Stop solution (Merck Millipore Corporation, catalog number: 109072 )
  Note: It is named “Sulfuric acid” on Merck Millipore Corporation website.
6. Wash buffer (see Recipes)
7. MDCK medium (see Recipes)
8. Virus diluent media (see Recipes)

Equipment

Haemacytometer (Hausser Scientific, catalog number: 1492 )
Multichannel pipette (Gilson, model: PIPETMAN Neo^® Multichannel )
Incubator, 37 °C, 5% CO₂ (Panasonic Corporation, Sanyo Electronics company, model: MCO-170AIC-PE )
SpectraMax M5 multi-detection microplate reader system (Molecular Devices, model: M5 )

Procedure

Prepare initial monoclonal antibody dilutions using virus diluent.
Add 50 μl of virus diluent to all wells in column 1 to 10.
Add 50 μl of the monoclonal antibody (1 mg/ml stock solution) to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette from the first well to each successive well from 1:1 to 1:128. Refer to Figure 1.
Discard 50 μl after the last dilution.

Figure 1. Serial dilution of test sample for MN assay. Add 50 μl of virus diluent to all wells in column 1 to 10 and add 50 μl of the sample to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette. Dilution factor of sample is 1 to 128.
Cover and hold in 37 °C, 5% CO₂ incubator while the diluted virus is being prepared. It is critical that the proper pH is maintained so that there will be no deleterious pH effects on the virus when it is added.
Dilute the virus in virus diluent to the correct working dilution as 100 TCID₅₀ /50 μl (TCID50: 50% Tissue culture infective dose, 100 TCID₅₀: 100x TCID₅₀).
Add 50 μl of diluted virus to wells containing antibodies and the virus control (VC) wells (A12, B12, C12 and D12) and do not add virus to the cell control (CC) wells (E12, F12, G12 and H12) and do not add virus to the column of wells reserved for the virus back titration.
Figure 2 showed detailed diagram for serial dilution of test antibody and back titration.

Figure 2. Serial dilution of antibody and back titration. Serial dilution of test sample (green box) and virus back titration (yellow box) is 2-fold dilution by 50 μl + 50 μl. Virus control (blue box) is also 50 μl + 50 μl, but cell control (red box) is only 100 μl of diluent.
Tap the plate gently to mix.
Add 100 μl of virus diluent to the CC wells.
Add 50 μl of virus diluent to the VC wells.
In each assay, include a back titration of the working dilution of virus because neutralizing antibody titer is changed sensitively depend on virus titer.
Add 50 μl of virus diluent to all wells in column 11.
Add 50 μl of the working dilution of virus to the first well and make 2-fold serial dilutions by transferring 50 μl using a multi-channel pipette from the first well to each successive well through well 12.
Discard 50 μl from well 12.
Add additional 50 μl of virus diluent to all wells for total volume to 100 μl.
Cover and incubate at 37 °C, 5% CO₂ for 1 h.
After 1 h, add 100 μl of diluted MDCK cells (which should be 70-95% monolayer and low passage <30) to each well of microtiter plates. Each well contains 1.5 x 10⁴ cells.
Incubate at 37 °C, 5% CO₂ for 18-20 h.
After incubation, remove the medium from microtiter plates and wash the plates with 200 μl of PBS and then remove PBS (overturn the plate by hand to spill the PBS and then swap remaining liquid using paper towel).
Add 100 μl of 80% cold acetone to each wells and incubate at room temperature (RT) for 10-12 min for fixation before ELISA.
Remove the acetone (overturn the plate by hand to spill the acetone and then swap remaining liquid using paper towel), let the plates air-dry for 10 min or until dry.
Dilute the primary antibody in antibody diluent to an optimum working dilution.
Wash the plate 3x with 300 μl of the wash buffer and add 100 μl of diluted primary antibody (1:1,000).
Cover and incubate for 1 h at RT.
Dilute the secondary antibody in antibody diluent to an optimum working dilution
Wash the plate 3x with 300 μl of the wash buffer and add 100 μl of diluted secondary antibody (1:2,000).
Cover and incubate for 1 h at RT.
Wash the plate 5x with 300 μl of the wash buffer and add 100 μl of freshly prepared TMB substrate.
Incubate at RT until the color change in the VC wells is intense and the corresponding color change in the CC wells in minimal.
Add 100 μl of stop solution.
Read the absorbance (O.D.) of wells at 490 nm using microtiter plate spectrophotometer.
The O.D. in the VC wells should be at least 0.8 and is typically in the range 1.0-1.5 though higher is acceptable.
The O.D. in the CC wells must ≤ 0.2.
The data are analyzed as follows.
1. 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 value that is used to calculate 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: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. In general, back titration titers of 16, 32, and 64 are acceptable.
  
  Example of back titration
  Median O.D. of CC = 0.2
  Median O.D. of VC= 1.0
  Mean of the VC median and the CC median = (0.2+1.0)/2= 0.6 = cut-off value
  O.D. of back titration wells (Table 1)
  
  Table 1. Example of O.D. of back titration wells
  Dilution
  1:1
  1:2
  1:4
  1:8
  1:16
  1:32
  1:64
  1:128
  O.D.
  2
  1.5
  1
  0.8
  0.5
  0.4
  0.3
  0.2
  
  1:16 (O.D.: 0.5) is the first well below the cut-off value (O.D. 0.6).
  Back titration titer is 1:16.
2. 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 antibody from the bottom, beginning at well H. Note the first well with an OD of less than the 50% cut-off. The reciprocal antibody dilution corresponding to that well is the 50% neutralization antibody titer for that antibody sample.
  
  Example of neutralizing antibody titer
  Median O.D. of CC = 0.2
  Median O.D. of VC = 1.0
  Mean of the VC median and the CC median = (0.2+1.0)/2 = 0.6 = cut-off value
  O.D. of back titration wells (Table 2)
  
  Table 2. Example of O.D. of neutralizing antibody wells
  Dilution
  1:1
  1:2
  1:4
  1:8
  1:16
  1:32
  1:64
  1:128
  O.D.
  0.1
  0.2
  0.3
  0.4
  0.5
  0.8
  1.5
  2.0
  
  1:4 (O.D.: 0.5) is the first well below the 50% cut-off value (O.D. 0.6).
  Neutralizing antibody titer is 1:16.

Recipes

Wash buffer
Phosphate buffered saline (PBS) with 0.3% Tween 20
MDCK medium
DMEM supplemented with
10% fetal bovine serum (FBS)
100 U/ml penicillin
100 mg/ml streptomycin
2 mM L-glutamine
Sterilize by filtration
Virus diluent media
DMEM, supplemented with 1% bovine serum albumin (BSA) (fraction V, protease free) (prepared as a 10% w/v solution in dH₂O, filter sterilized, and stored at 4-8 °C)
100 U/ml penicillin
100 mg/ml streptomycin
20 mM HEPES
Prepare fresh for each assay
Sterilized by filtration

Acknowledgments

We thank Dr. Jun Myung Kim and Dr. Sang Hoon Han for providing blood samples from Severance Hospital, and Dr. Yasuo Watanabe of SC World, Inc. for assistance in ISAAC assays for antibody screening. This study was supported by a grant of the Korea Healthcare technology R&D Project‚ Ministry of Health&Welfare‚ Republic of Korea. (Grant No. A103001), the National Natural Science Foundation of China (NSFC, Grant No. 81401671), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB08020100), the China National Grand S&T Special Project (Grant No. 2015ZX09304005) and G. F. G. is a leading principal investigator of the NSFC Innovative Research Group (Grant No. 81321063). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

References

Klimov, A., Balish, A., Veguilla, V., Sun, H., Schiffer, J., Lu, X., Katz, J. M. and Hancock, K. (2012). Influenza virus titration, antigenic characterization, and serological methods for antibody detection. Methods Mol Biol 865: 25-51.
Manual for the laboratory diagnosis and virological surveillance of influenza. WHO global influenza surveillance network.