Serum tube

The method used a well-established immunogenicity model previously reported.^{12 (link)} Briefly, BALB/c mice (Mus musculus, females, 6 to 8 weeks) purchased from Charles River Laboratories (Wilmington, MA, USA) were housed in microisolator units. Mice (six per group, as determined by power analysis for 85% power) were vaccinated with rNA in the presence or absence of squalene-based oil-in-water emulsion adjuvant, AF03 (Sanofi, 5% squalene). The rNA from samples recovered from thermal stress studies was diluted in NaPBS or KP-10% to 40 μg/mL and then mixed with AF03 adjuvant at 1:1 ratio (25 µL vaccine:25 µL AF03) resulting in a final concentration of 2.5% squalene. Mixture was administered via intramuscular injection in 50 µL volume (1 μg rNA/dose/animal) on day 0 (D0) and then boosted with the same dose on day 21 (D21). Blood was collected on day 35 by cardiac puncture into serum tube containing clot activator and separator gel (Sarstedt, Nümbrecht, Germany) and tested for presence of NA-specific antibodies by ELLA.