Evaluating Anti-influenza Antibodies

Serum anti-influenza levels were evaluated using peptides or live/inactivated influenza viruses, using the procedure previously described [14 (link)]. Briefly, 96-well NUNC MaxiSorp ELISA plates (Fisher Scientific, Pittsburg, PA, USA) were coated with peptides (1 µg/mL) or influenza viruses (10⁵ TCID₅₀ per mL) in PBS (100 µL per well) for 18–24 h at room temperature (RT) and 2–8 °C, respectively. Serial dilutions of serum samples starting at 1:100, with five-fold dilutions (1:100, 1:500, 1:2500, 1:12,500, etc.), were added to antigen-coated plates blocked with 3% normal goat serum (NGS, Southern Biotech, Birmingham, AL, USA) and detected with Horse Radish Peroxidase (HRP)-conjugated isotype-specific goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA). TMB Substrate Solution (Fisher Scientific, Pittsburg, PA, USA) was added prior to being quenched with TMB STOP solution (Fisher Scientific, Pittsburg, PA, USA). The absorbance values (450 nm) of each well were obtained using a SpectraMax Plus Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Data were expressed as endpoint titers defined as the reciprocal of highest dilution that yielded an absorbance value greater than or equal to twice that of the pooled pre-immune serum. Due to a minimum starting serum dilution of 1:100, titers below 100 were reported as nil.