Plasma Antibody Quantification

Plasma was separated from whole blood collected from mice using saphenous bleeding, by centrifugation at 2000 g for 10 min. Mouse plasma samples were collected at 2, 4, 8, 9, 10, 12, 14, and 16 weeks following vaccination, and the concentrations of PCS5-specific plasma IgG antibodies were quantified using a previously published protocol.³⁹ Briefly, 20 μg of PCS5 was coupled to 1.25 × 10⁶ Bio-Plex Pro Magnetic COOH Beads using a Bio-Plex Amine Coupling Kit. 50 μL of plasma (1:80 diluted) were incubated with 2,500 beads. SIV-specific IgG was detected with phycoerythrin-labeled mouse antimonkey IgG at 5 μg/mL. Bead fluorescence intensities were acquired on the Bio-Plex 200 system and converted to concentrations as previously described.³⁹