Streptavidin hrp

p24 ELISA was performed as described previously^{79 (link)}. Briefly, virus-pulsed cells and supernatant containing viral particles were lysed in ELISA lysis buffer (0.05% Tween 20, 0.5% Triton X-100, 0.5% casein in PBS). Anti-HIV-1CAp24 antibody (1:1000 or 1:2000; clone 183-H12-5C; NIH AIDS Research and Reference Reagent Program, Cat# 3537) was bound to Nunc MaxiSorp plates overnight. Lysed samples were captured for 2 h and incubated with biotinylated antibody to HIV-1CAp24 (1:2000 or 1:4000; clone 31-90-25; ATCC, Cat# HB-9725). 31-90-25 was biotinylated with the EZ-Link Micro Sulfo-NHS-Biotinylation Kit (Pierce). Samples were detected using streptavidin-HRP (Fitzgerald) and 3,3^′,5,5^′-tetramethylbenzidine substrate (Sigma-Aldrich). CAp24 concentrations were determined using recombinant HIV-1 IIIB p24 recombinant protein for standards (NIH AIDS Research and Reference Reagent Program).

Supernatant containing viral particles was lysed in Triton X lysis buffer (0.05% Tween 20, 0.5% Triton X-100, 0.5% casein in PBS). Gag p24 antibody (clone 183-H12-5C, AIDS Reagent Program cat# 1519 from Dr. Bruce Cheseboro and Dr. Hardy Chen) was bound to Nunc MaxiSorp plates (ThermoFisher cat# 12-565-135) at 4°C overnight. Lysed samples were captured for 2 hr and then incubated with biotinylated antibody to Gag p24 (clone 31-90-25, ATCC cat# HB-9725) for 1 hr. Clone 31-90-25 was biotinylated with the EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher cat# PI-21925). Clones 31-90-25 and 182-H12-5C were purified using Protein G columns (GE Healthcare, cat# 45-000-054) following the manufacturer’s instructions. Samples were detected using streptavidin-HRP (Fitzgerald, Acton, Massachusetts) and 3,3′,5,5′-tetramethylbenzidine substrate (Sigma cat# T8665-IL). CAp24 concentrations were measured by comparison to recombinant CAp24 standards (cat# 00177 V, ViroGen, Watertown, Massachusetts).