SIV-Specific Antibody Quantification via ELISA

SIV-Gag and Env-specific IgG were detected in plasma using ELISA as previously described [39 (link)]. In brief, purified SIV Gag or Env antigens (Immunodx, Woburn, MA, USA) were used as coating antigen. After washing, serial four-fold dilutions of plasma in blocking buffer were added in wells. Following washing, plates were further incubated with peroxidase-conjugated goat anti-monkey IgG secondary antibodies (Exalpha Biologicals, Shirley, MA, USA) diluted in blocking buffer. After washing, all wells were further incubated with O-Phenylenediamine Dihydrochloride substrate (Sigma-Aldrich, St. Louis, MO, USA), color was developed, and reaction was stopped by adding 4N sulfuric acid. The plate was read at an optical density (OD) of 490nm using Elx800 reader (Biotek, Winooski, VT, USA). All samples were assayed in duplicates with appropriate positive and negative controls. For quantification of SIVGag or Env specific IgG responses, rhesus IgG (NIH-NHP Reagent Resource) standard was used. Nonlinear regression using a sigmoidal dose-response variable slope model was used to interpolate concentrations from the standard curve as reported earlier [39 (link)].