Alkaline phosphatase conjugated goat anti human fab secondary antibody

Antibodies b12, 2G12, PGT121, PGT128 and PGT135 were obtained from the International AIDS Vaccine Initiative (IAVI, New York, NY). Microtitre ELISA plates (Corning) were coated with mouse anti-HIS capture antibody (2 μg mL⁻¹ in PBS; Life Technologies) overnight at 4 °C. Plates were washed five times with a solution of PBS containing 0.05% Tween 20 (v/v) and then blocked for 1 h at room temperature with 5% non-fat milk in PBS + 0.05% Tween (blocking buffer). Gp120 was then added at a concentration of 2 μg ml⁻¹ in blocking buffer, followed by incubation for 1 h at room temperature. Plates were washed (×5) before addition of primary antibodies (b12, 17b, 2G12, PGT121, PGT128 & PGT135). A 1:5 dilution series was used starting at 20 μg ml⁻¹ or 40 μg ml⁻¹ in blocking buffer. Incubation was carried out at room temperature for 2 h. Plates were then washed (×5) and alkaline phosphatase-conjugated goat anti-human Fab secondary antibody (Thermo Scientific Pierce) was added as a 1:1000 dilution in blocking buffer. Plates were washed (×5) and then AP substrate (50 μL per well) was added. Once color had developed the OD at 405 nm was measured.