At predetermined time intervals the release medium was sampled (3 ml) and replaced with fresh pre-warmed dissolution media. Samples were diluted in PBS-T for concentration analysis by ELISA. For rods dissolution volume was 20 ml and sample volume was 2 ml. Dissolution volumes were selected to maintain sink
conditions. Stability assessment was carried out in a similar fashion to the described release Dorsomorphin protocol. Following complete dissolution of the CN54gp140 containing lyophilized solid dosage tablets in PBS-T (30 ml) a sample was taken and diluted in PBS-T for concentration analysis by ELISA. Animals were assigned to experimental groups where n = 5 ( Table 1). Mice received a subcutaneous (s.c.) prime (Day 0) then an intra-vaginal (i.vag.) boost three times at 21-day intervals (Days 21, 42, 63) with vaginally administered rod formulations
( Table 1). Mice were lightly anesthetised and the rod formulations were inserted into the vagina using a positive displacement pipette (Gilson Microman – 100 μl maximum volume) and a tip with the end cut off and filed down to smoothness. To thin the vaginal epithelium and improve protein uptake, mice were treated subcutaneously with Selleck ZVADFMK 2 mg of depoprovera (in 50 μl PBS) 5 days prior to the first and third vaginal immunization. Blood samples were taken from the tail vein of mice on Days 20, 41, 62, and 83 and by cardiac puncture on Day 120. Blood samples were centrifuged following clotting for collection of sera. Vaginal lavages were else conducted on Day 83. Vaginal lavages were collected and pooled by flushing the vaginal lumen three
times with a 25 μl volume of PBS using a positive displacement pipette. 5 μl of 25X protease inhibitor cocktail was added to the vaginal eluates, which were incubated on ice for 30 min prior to centrifugation to remove the mucus/cellular pellet. All samples were stored at −80 °C until analysis. Binding antibodies against CN54gp140 in vaginal lavage and serum samples were measured a quantitative ELISA. 96-Well plates were coated with CN54gp140 and blocked with 1% BSA as before. IgG or IgA standards were used on each plate to quantify the CN54gp140 specific antibodies. Experimental samples were diluted 1:100, 1:1000 and 1:10,000 (sera) or 1:10 and 1:50 (lavage) to ensure the absorbance reading measured fell within the linear range of the standard curve. Bound IgG was detected by incubation for 1 h at 37 °C with HRP-conjugated goat anti-mouse IgG, bound IgA was detected using biotinylated anti-mouse IgA and followed by Streptavidin-HRP. Plates were washed and developed with 50 μl TMB/E substrate and the reaction was terminated by the addition of 50 μl of 2 M H2SO4 and read at A450. Vaginal lavage values were normalised against the total IgA or IgG measured in the same sample. Semi-solids (Table 2) were prepared using either an overhead stirrer or HiVac® bowl, the choice of which was dependent upon the viscosity of the systems being prepared.