gp140 standards and samples were added to the wells and incubated for 2 h at 37 °C. Detection of gp140 was performed by incubation

for 1 h at 37 °C with 2 μg/ml 5F3 anti-gp140 human mAb in Buffer 2 (PBS supplemented with 2% skimmed powder milk, 5% porcine serum and 0.5% Tween-20), followed by incubation for 1 h at 37 °C with goat anti-human IgG-HRP (SouthernBiotech) in Buffer 2. Plates were developed with TMB for 20 min in the dark. The reaction was stopped with 1.0 N H2SO4 and O.D. read at 450 nm. Human cytokines/chemokines in cell culture supernatants were detected using an in-house multiplex assay following a protocol recommended by the manufacturers (R&D) as previously described [24]. Female Balb/c mice, 6–8 week old, were obtained from Harlan Olac Ltd., UK. Mice were kept at the Biological Research Facility, St. George’s University of London. All check details procedures were performed in accordance with the United Kingdom’s Home Office standards under the Animals Scientific Procedures Act, 1986, and approved by the School’s Ethical Review Committee. Mice were inoculated i.d. with 12.5 μg (TT) or 20 μg (gp140) in a total volume of 100 μl

in sterile saline on both dorsal flanks following a prime-boost-boost protocol at 4 (TT) and 3 (gp140) week intervals. For i.n. immunization, 20 μg gp140 with or without NP in a maximum volume of 25 μl were gently dispensed in the animal’s nostrils after isofluorane-induced anaesthesia. Antigen-adsorbed NP were prepared the same day of immunization. Fresh components of the formulations were used in these experiments selleck products found because they were performed in parallel

with the NP colloidal stability studies (see Fig. 1B). These studies suggested nonetheless that similar results would be obtained using the same formulation over time. Alum-Ag complex was prepared by mixing equal volumes of Ag and Alum solution (Imject Alum, Pierce, Rockford, IL), and mixed by rotation for 30 min at room temperature. Blood samples were collected before priming, 1–3 days before boosting, and at 4 (TT) and 3 (gp140) weeks after the last boost. Serum was separated from clotted blood and stored at −80 °C until further use. Vaginal samples were collected by flushing 30 μl of PBS three times into the vagina of anaesthetized animals, pooled and supplemented with 8 μl of a 25× protease inhibitor cocktail (Roche Diagnostics, Manheim, Germany). Samples were incubated for 30 min on ice and then spun at 14,000 rpm for 10 min. Supernatants were collected and stored at −80 °C. Eight fecal pellets/mouse were collected, weighed and mixed with 4× their weight of 1× protease inhibitor cocktail. Samples were homogenized to dissolve the pellets and incubated on ice for 1 h. The samples were spun twice at 14,000 rpm for 10 min, and cleared supernatants stored at −80 °C. Nasal samples were obtained after sacrifice of the animals by flushing the nasal cavity with 300 μl of PBS containing 1× protease inhibitor cocktail.