Env- and Gag-specific rectal swab IgA titers measured by Bio-Plex were also compared to ELISA data. the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (DNA prime+SIVmac239-recombinant adenovirus boost regimen. All RMs, excluding the SIV-naive control animals, HJC0350 were challenged with low-dose SIVmac239 by the intrarectal route and confirmed to be infected by a positive SIVmac239 viral load. All samples assayed herein were obtained between 7 and 123 days postinfection. Sera were obtained in 2004C2005 or in 2012 and stored at ?80C. Rectal swabs were obtained in 2011C2012 and stored at ?80C. Viral loads were determined within 20 days of sampling and were found to be between 103 and 108 genome copy equivalents per milliliter. Rectal swabs HJC0350 Rectal swabs were Spry3 collected as previously described using Weck-Cel? Eye Spears (Beaver-Visitec, Waltham, MA).37 Sample collection minimized bleeding and subsequent elution was performed according to the published protocol.37 Prior to use, samples were tested for the presence of blood using Hemoccult? Test Cards (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol using HJC0350 20?L of eluate. If blood was detected, samples were not used for this study. Bead coupling One milliliter of Bio-Plex COOH (carboxylated) beads were conjugated to SIV proteins according to the manufacturer’s protocol using the Bio-Plex amine coupling kit (Bio-Rad). Beads were counted just prior to conjugation using a Vi-Cell Viability Analyzer (Beckman-Coulter) to ensure a consistent bead-to-protein ratio since bead loss occurs during the conjugation process.38 Protein was added at a ratio of 25?g of protein/5106 beads for Envelope gp130 (produced in-house), Gag p55 (Protein Sciences Corp., Meriden, CT), and Pol (Immune Technology, New York, NY), or 75?g/5106 beads for Nef (Immune Technology). Final volume was adjusted to 5?mL in phosphate-buffered saline (PBS), and tubes HJC0350 were agitated for 2?h. Beads were then washed with 5?mL of PBS, resuspended in 2.5?mL of Stabilguard blocking buffer (SurModics, Eden Prairie, MN), and agitated for 30?min.39 Beads were washed, resuspended in 400?L of storage buffer, counted, aliquoted, and stored at ?80C. ELISA Polystyrene, flat-bottom, high-binding, half-area plates (Corning, Kennebunk, ME) were coated overnight at 4C by adding 17.5?ng of Env gp130, 125?ng of Gag p55 (IgG ELISA), 150?ng of Env gp130 or Gag p55 (IgA ELISA) per well. Plates were washed in PBS/0.02% Tween-20 and blocked for 1?h at 37C with 120?L of PBS/3% bovine serum albumin (BSA; IgG ELISA) or PBS/3% milk (IgA ELISA). Plates were washed, and serum or swab eluate was added HJC0350 at 1:100 or 1:10 diluted in PBS/1% BSA (IgG ELISA) or PBS/1% milk (IgA ELISA), respectively. Serum was diluted threefold across the plate, while swab eluate was diluted twofold. Plates were incubated and washed as above before the addition of biotin-conjugated goat antimonkey IgG for serum (0.125?g/mL in PBS/1% BSA; Rockland Immunochemicals, Gilbertsville, PA) or goat antimonkey IgA for swabs (0.66?g/mL for Env ELISA, 4?g/mL for Gag ELISA, in PBS/1% milk; Rockland Immunochemicals). Plates were incubated and washed as before prior to the addition of streptavidinChorse radish peroxidase (1?g/mL; Biolegend, San Diego, CA). After incubation and washing, TMB Substrate Reagent Set (3,3,5,5-tetramethyl benzidine; Biolegend) was added according to the manufacturer’s protocol. The reaction was stopped with 2?N H2SO4 and plates were read at 450?nm using a VERSAmax ELISA reader (Molecular Devices, Sunnyvale, CA). Bio-Plex assay Analysis of serum and swab eluate with Bio-Plex assays were performed using conditions suggested by the manufacturer (Bio-Rad). One hundred fifty microliters of assay buffer (PBS, 0.5% casein, 0.1% BSA, 0.02% Tween-20, 0.05% sodium azide, pH 7.4) was added to each well of a Multiscreen HTS,.