For HIV-1 Gag p24 determination, two days after transfection, p24 antigen in cell culture media was detected using HIV Type 1 p24 Antigen ELISA 2.0 (ZeptoMetrix) following kit instructions.
For plasma antibody determination, recombinant HIV-1 p24 protein (Abcam, 0.5 μg/ml) was coated in 96-well EIA/RIA plates overnight at 4 °C. The plates were blocked with 5% skim milk at 37 °C for 1 h. After washing, plasmas (two-fold serially diluted from 1:50 or 50-fold diluted) were added and incubated for 1 h at 37 oC. After washing, goat anti-mouse IgG H&L (HRP) secondary antibody (Abcam) diluted 1:50,000 was added. Plates were then incubated at 37 oC for 1 h. After extensive washes, 100 µl of the substrate was added and incubated for 10 min at room temperature, followed by adding 100 µl of stop solution. The optical density at 450 nm was determined using a Varioskan LUX (Thermo Scientific).
For plasma antibody determination, recombinant HIV-1 p24 protein (Abcam, 0.5 μg/ml) was coated in 96-well EIA/RIA plates overnight at 4 °C. The plates were blocked with 5% skim milk at 37 °C for 1 h. After washing, plasmas (two-fold serially diluted from 1:50 or 50-fold diluted) were added and incubated for 1 h at 37 oC. After washing, goat anti-mouse IgG H&L (HRP) secondary antibody (Abcam) diluted 1:50,000 was added. Plates were then incubated at 37 oC for 1 h. After extensive washes, 100 µl of the substrate was added and incubated for 10 min at room temperature, followed by adding 100 µl of stop solution. The optical density at 450 nm was determined using a Varioskan LUX (Thermo Scientific).