Acidic stop solution

Antibody responses against p27 and gp41 were quantitated by regular ELISA using SIVmac239 p27 purified recombinant protein (Catalog# 13446; obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) and recombinant SIV gp41 (Catalog# 5019; ImmunoDX) to coat plates at 10 μg/ml in PBS. Plates were then washed, blocked and incubated for 1 h at 37°C with a 1:20 dilution of the corresponding monkey sera or 5L7 antibody diluted to 2 μg/ml. Antibodies binding the p27 antigen or the gp41 were detected with a goat anti-monkey secondary antibody (Catalog# 2015-05, Santa Cruz) and developed with TMB High Sensitivity Substrate Solution (Catalog# 421501, Biolegend). The reaction was stopped by the addition of 50 μl of acidic stop solution (SouthernBiotech, Birmingham, AL), and the optical density at 450 nm was measured using a Wallac Victor plate reader (Perkin-Elmer, Waltham, MA).

PepScan or ELISA against a panel of 218 peptides overlapping the entire SIVmac239 envelope protein was used to detect antibody responses to the viral spike. Fifteen-mer-length peptides, each successive peptide overlapped by 11 amino acids, were obtained from the NIH AIDS Research and Reference Reagent Program. Peptides were properly resuspended and used to coat ELISA plates at 40 μg/ml in phosphate-buffered saline (PBS). Plates were then washed, blocked and incubated for 1 h at 37°C with a 1:20 dilution of the corresponding monkey sera or 5L7 antibody diluted to 2 μg/ml. Binding antibodies were detected with an HRP-conjugated goat anti-human IgG antibody (SouthernBiotech) diluted 1:1,000 in 5% non-fat powdered milk in PBS and developed with soluble tetramethylbenzidine (TMB) reagent (Calbiochem, Gibbstown, NJ). The reaction was stopped by the addition of 50 μl of acidic stop solution (SouthernBiotech, Birmingham, AL), and the optical density at 450 nm was measured using a Wallac Victor plate reader (Perkin-Elmer, Waltham, MA).