Mouse anti his tag mab

D11A.B5, D11A.F2, and D11A.F9 were biotinylated using EZ-Link NHS-Biotin (Pierce Biotechnology, Thermo Scientific) per the manufacturer s protocol. For cross-competition ELISA assays, 16055 NFL TD CC trimers were captured on the ELISA plate by a mouse anti-His tag mAb (R&D Systems) coated at 2 μg/ml in PBS at 4°C overnight. Five-fold serial dilutions of various bNAbs and non-bNAbs were pre-incubated with the captured trimer at RT for 30 min prior to addition of the biotinylated mAbs at a concentration previously determined to give ~75% of the maximum binding signal (i.e. binding to trimer with no competitor present) for 60 min at RT. The bound biotinylated mAbs were detected using HRP-conjugated streptavidin (Sigma). The signal was detected with TMB substrate and reaction stopped with 1 N sulfuric acid as above. Wells were blocked with 5% non-fat milk in PBS-T (0.2% Tween-20) after coating with the anti-His mAb and washed with PBS-T after each incubation step. Samples were diluted in 10% blocking buffer. Competition is expressed as percent inhibition where 0% was the absorbance measured with no inhibitor present.

HIV-1 Env-specific IgG titers in plasma were measured by ELISA as previously described (Ingale et al., 2016 (link)). MaxiSorp 96-well plates (Nalgene Nunc International) were coated overnight at 4°C with a mouse anti-His tag mAb (1.5 μg/ml; R&D Systems). After blocking the plates with PBS containing 2% of bovine serum albumin for 2hr at RT, the plates were incubated with 16055 NFL TD CC at 3 μg/ml for 2 hr at RT. Next, the plates were incubated with 5-fold serial dilutions of plasma starting at 1:50 for 1hr at RT. Env-specific IgG was detected by adding a secondary horseradish peroxidase (HRP) conjugated anti-monkey IgG antibody (1:10000) (Nordic Immunology, Tillburg, The Netherlands) and the signal developed by addition of tetramethylbenzidine (TMB) substrate (Invitrogen). Adding equal volume of 1M H₂SO₄ stopped the reaction and the optical density (OD) was read at 450 nm. Between each incubation step, the plates were washed 6 times with PBS supplemented with 0.05% Tween 20. The half-max binding titers (OD₅₀) for each sample was calculated by interpolation from mean OD₅₀ values calculated from an immunized plasma using the formula (ODmax-ODmin)/2).