Western blots were performed by incubating gp140 and gp140ΔV2 at 0.3 mg/ml with 50–69 and 2F5 antibodies (NIH AIDS Reagent Program) at 2 μg/ml, transferring to PVDF membrane overnight, blocking with 5% milk for 3 h, then washing in 0.1 M PBS-T 3X. The gp140-2F5, gp140ΔV2-2F5, gp140-50-69, and gp140ΔV2-50-69 complexes were then incubated in anti-human alkaline phosphatase-conjugated secondary antibody (Life Technologies, Grand Island, NY) diluted 1:500 for 1 h, washed in 0.1 M PBS-T, and the membranes were developed in SIGMAFAST™ BCIP®/NBT (Sigma-Aldrich, St. Louis, MO).
ELISAs were conducted on 96-well plates by adsorbing gp140 at 0.3 mg/ml, incubating with 50–69 and 2F5 at 2 μg/ml, applying anti-human alkaline phosphatase-conjugated secondary antibody (Life Technologies, Grand Island, NY) diluted 1:500, then adding PnPP substrate (Sigma-Aldrich, St. Louis, MO). Plate readings were done at 405 nm.
ELISAs were conducted on 96-well plates by adsorbing gp140 at 0.3 mg/ml, incubating with 50–69 and 2F5 at 2 μg/ml, applying anti-human alkaline phosphatase-conjugated secondary antibody (Life Technologies, Grand Island, NY) diluted 1:500, then adding PnPP substrate (Sigma-Aldrich, St. Louis, MO). Plate readings were done at 405 nm.