Procedures

Procedure 1: Cell lines and influenza A virus (H1N1) generation

Definitions & Abbreviations: IAV # Influenza A virus

Reagents and solutions

• HEK293T cells (ATCC, CRL-3216, Manassas, VA, USA)
• MDCK cells (ATCC, CCL-34, Manassas, VA, USA)
• Influenza A/CA/04/09 (H1N1) virus

Steps

1. Influenza A/CA/04/09 (H1N1) virus is generated by multi-plasmid transfection of HEK293T cells followed by a 48 hour amplification and titration in MDCK cell as described by Neumann et al. (2012) ('Reverse Genetics of Influenza Viruses' in Kawaoka, Y. and Neumann, G. (eds.) Influenza Virus: Methods and Protocols. 2012 Humana Press, pp. 193-206.)

Procedure 2: Influenza A virus (H1N1) infection and blood collection

Reagents and solutions

• Isoflurane
• PBS

Steps

1. Mice are lightly anesthetized with isoflurane inhalation and infected intranasally with either 5000 focus forming units (FFU) of influenza A/CA/04/09 in 50 μL PBS. Approximately 3 mice per F1 per time point are infected with IAV, and animals are monitored daily.
2. At 7, 10, 15, 29 or 45 days post-infection mice are euthanized by isoflurane overdose and bilateral thoracotomy; blood is collected by terminal bleed and the sera aliquoted and stored at -80°C.

Procedure 3: Antibody measurement

Equipment, software, and supplies

• Spectrophotometer

Reagents and solutions

• HA antigen (BEI Resources, NR13691, Manassas, VA, USA)
• Carbonate buffer, composition 0.32M Na₂CO₃, 0.68M NaHCO₃
• Wash buffer, 0.033% Tween-20 in PBS
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM, RRID: AB_2794201 (Southern Biotech, 1020-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1, RRID: AB_2650509 (Southern Biotech, 1070-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2a, RRID: AB_2734756 (Southern Biotech, 1080-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2c, RRID: AB_2794466 (Southern Biotech, 1079-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2b, RRID: AB_2794521 (Southern Biotech, 1090-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG3, RRID: AB_2794573 (Southern Biotech, 1100-05, Birmingham, AL, USA)
• Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, RRID: AB_2619742 (Southern Biotech, 1030-05, Birmingham, AL, USA)
• Citrate substrate, composition 0.05M sodium citrate, 0.05M citric acid, 1 mg/mL OPD, and 0.216% hydrogen peroxide
• Sodium fluoride, (0.1M)

Steps

1. IAV hemagglutinin (HA)-specific antibody are quantified by ELISA. 96-well flat-bottomed plates are coated with HA antigen (diluted in carbonate buffer to 1 µg/mL). Half-log serum dilutions (10² to 10^5.5) are prepared in wash buffer, added to the coated plates, and then incubated overnight in a humidified chamber set at a temperature of 4°C.
2. Plates are washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP conjugated goat anti-mouse, IgM, IgG1, IgG2a pooled with IgG2c (i.e. IgG2ac), IgG2b, IgG3, Total IgG) for approximately 2 hr at 4°C. IAV plates are washed and developed in the dark for 30 min at room temperature with citrate substrate, and then stopped with sodium fluoride and read immediately at a wavelength of 450 nm.
3. Area under the curve (AUC) values for each sample is calculated by subtracting background OD measurements set against dilution factors. AUC was found to be the most inclusive measure that captured the dynamic range of antibody levels across the sample set, and was also found to perform better than other measurements such as half maximal or lowest positive titer.