SIV gp120 Linear Peptide ELISA

Plasma samples were assayed by PEPSCAN analysis using SIV_mac251 gp120 linear peptides^{16 (link)}. ELISA plates (Nunc Maxisorp) were coated with 100 ng of each of the 1–89 overlapping peptides (with 15 amino acids each encompassing the entire SIV_mac251 gp120 sequence) in 50 mM NaHCO3, pH 9.6, per well, incubated overnight at 4 °C, and blocked with 200 μl of Pierce SuperBlock blocking buffer in PBS for 1 h at room temperature. Serum samples were diluted at 1:50 in sample diluent (Avioq), and 100 μl were added to the plate and incubated for 1 h at 37 °C. Plates were washed six times with PBS Tween 20 (0.05%) and incubated with 100 μl anti-human HRP diluted at 1:120,000 in sample diluent (Avioq) to all wells and incubated, covered, for 1 h at 37 °C. The plates were again washed six times and developed using 100 μl of K-Blue Aqueous substrate (Neogen) to all wells and incubated 30 min at room temperature. The reaction was stopped by adding 100 μl of 2 N sulfuric acid to all wells and plate was read at 450 nm on a Molecular Devices E-max plate reader.