Materials and Reagents

1. 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 )
   
   
2. 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

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

Procedure