Alp goat anti mouse igg

TT-specific ASC were enumerated by ELISPOT, as previously described (15 (link), 19 (link), 20 (link), 38 (link)). MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 10 μg/ml TT overnight at 37°C, blocked with complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.). Duplicates of cells from spleen and bone marrow in four three-fold dilutions starting with 1 × 10⁷ cells in 100 μL in complete RPMI 1640 per well were incubated for 5 hours at 37°C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4°C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). The number of spots, each representing a cell secreting TT-specific IgG antibodies, was counted with ELISPOT reader ImmunoSpot^® S6 ULTIMATE using ImmunoSpot^® SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).

PPS1- and TT-specific ASC were enumerated by ELISPOT, as previously described (9 (link), 48 (link)). MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 20 μg/ml PPS1 or 10 μg/ml TT overnight at 37°C, blocked with complete RPMI 1640 (Life Technologies BRL, Life Technologies, Paisley, U.K.). Duplicates of cells from spleen and BM were tested in four three-fold dilutions starting with 1 × 10⁷ cells in 100 μL in complete RPMI 1640 per well (9 (link), 48 (link)) and incubated for 5 h at 37°C, washed and incubated with ALP-goat anti-mouse IgG (Southern Biotechnology Associates) overnight at 4°C, and developed by 5-bromo-4-chloro-3-indolylphosphate and NBT in AP development buffer (Bio-Rad Labs, Hercules, CA). The number of spots, each representing a cell secreting specific Abs, were counted by ELISPOT reader ImmunoSpot^® S6 ULTIMATE using ImmunoSpot^® SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).