Quantification of Mucosal Antibodies

Saliva was eluted from sponges as described previously (69 (link)). For analysis of intestinal Abs, 2 to 3 g of freshly collected stool was added to 5 ml of sterile 1× phosphate-buffered saline (PBS) plus 0.5% bovine serum albumin supplemented with 50 μl of 100× protease inhibitor cocktail (Sigma-Aldrich, Corp., St. Louis, MO) and snap-frozen. Clarified fecal extracts were prepared as previously described (70 (link)) and concentrated to approximately 0.5 ml with an Amicon Ultra-4 50K centrifugal filter unit (Millipore, Billerica, MA).
1086.C gp120 IgG and IgA Ab levels in fecal extracts and saliva were measured by BAMA with gp120-conjugated Bio-Plex magnetic beads (Bio-Rad Laboratories, Inc., Hercules, CA) as previously described (71 (link)). Total IgG or IgA concentrations in secretions were measured by enzyme-linked immunosorbent assay (ELISA) with previously calibrated normal monkey serum as the reference standard (72 (link)). Anti-gp120 IgG or IgA concentrations measured in each secretion were divided by the total IgG or IgA concentration to obtain the specific activity (nanograms of IgG or IgA per microgram of total IgG/IgA). The specific activity was considered significant if it was greater than the mean activity measured in naive-infant control samples plus 3 standard deviations.