Titermax adjuvant

Six week old BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were immunized with 9 μg of A/chicken/Hong Kong/G9/1997 (H9) HA mixed with TiterMax adjuvant (Sigma-Aldrich, St. Louis, MO), followed by 1 boost of 9 μg of A/Vietnam/1203/2004 (H5) HA mixed with TiterMax adjuvant, and 1 boost of 9 μg of A/Singapore/1/1957 (H2) HA mixed with TiterMax adjuvant by subcutaneous injection, at 2-week intervals. Two weeks after the final boost, mice were anesthetized and challenged intranasally with 5 50% lethal dose (LD₅₀) of A/Puerto Rico/8/1934 virus. Body weights were monitored daily and mice that reached a study endpoint of 25% loss in body weight were humanely euthanized using CO₂. In passive MAb transfer experiments, mice were administered intraperitoneally with MAbs 6 h before challenge with 5 LD₅₀ of A/Puerto Rico/8/1934 virus.

The recombinant protein LmxMBA (rLmxMBA) was obtained as described above, and female BALB/c mice were immunized with 10 μg/dose. Animals received only two intraperitoneal (i.p.) doses with 15 days of interval; the immunization doses were administrated in TiterMax adjuvant (1 : 1 mixture) (Sigma, St. Louis, MO, USA), and 30 days after the immunization scheme, mice were bled by cardiac puncture to obtain immune sera.

To generate bacterial expression vectors for GST fusion proteins, partial cDNA fragments encoding AsHSF3 (amino acids 300–453) and XtHSF3 (amino acids 300–553) were inserted into a pGEX-2T vector (GE Healthcare). Recombinant GST fusion proteins were expressed in Escherichia coli by incubating with 0.4 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) at 37°C for 3 h. Bacterial lysates were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the respective fusion proteins were excised, electroeluted, and concentrated using Amicon ultra-4 centrifugal filter units (EMD Millipore). The proteins were emulsified with an equal volume of TiterMax adjuvant (Sigma-Aldrich) to immunize rabbits. Blood samples were collected from rabbit marginal ear veins using 18-gauge needles, and were allowed to clot for 60 min at 37°C. The clots were placed at 4°C overnight to allow them to contract. The antisera for AsHSF1 (anti-AsHSF3-1) and XtHSF3 (anti-XtHSF3-2) were extracted from the clots by centrifugation at 10,000 g for 10 min at 4°C, and were stored at -80°C after the addition of sodium azide to 0.02%.