ELISA for Evaluating HIV-1 Env Peptide Binding

Enzyme-linked immunosorbent assays (ELISA) were performed as previously described^{8 (link)}. 384-well ELISA plates (Corning Life Sciences) were coated with TNC proteins (TNC-S, TNC-L, fn 1–8, fn 6–8, fn A-D, fbg, FNall, FNall+fbg) (Figure 1A), human albumin, or bovine serum albumin at 100 μg/ml in 0.1M NaHCO₃ and incubated overnight at 4°C. Blocking was performed using Super Block (1X PBS, 4% whey protein, 15% goat serum, 0.5% Tween 20) for 1 hour at RT. HIV-1 Env peptides (including MN.3 gp120, MN.3 gp70 V3, and the linear peptides MN.V3, MN.V3 15-mer, MN.V3 321/322, MN.V3 326/327, and MN.V3 321/322/326/327) were titrated using a two-fold serial dilution in Super Block starting at 500 μg/ml for the linear peptides, 1,280 μg/mL for MN.3 gp120, and 1,000 μg/ml for MN.3 gp70 V3. An HIV-1 gp120 specific monoclonal antibody (CH22 or 16H3) was used as a primary antibody while detection was done using goat anti-human or anti-mouse HRP labeled (1:5,000) as a secondary antibody. Signal was then detected with SureBlue Reserve 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (VWR) and absorbance recorded at 450nm using a Spectramax Plus spectrophotometer (Molecular Devices).