5 bromo 4 chloro 3 indolylphosphate

T-cell immunity to HIV was assessed in purified PBMC by ELISpot assay (1 × 10⁶ PBMC/well) using an overlapping peptide (OLP) set of 410 OLP as described previously (37 (link)). In brief, 96-well ELISpot plates (Millipore) were coated with mouse anti-human IFN-γ antibody 1-D1k (Mabtech) and incubated overnight. The next day, plates were washed six times with phosphate-buffered saline (PBS), and PBMC were added at 100,000 cells/well along with peptide at a final concentration of 2 μg/ml. After incubation for 16 h at 37°C in 5% CO₂, plates were washed 6 times with PBS and the mouse anti-human IFN-γ-biotinylated 7-B6-1 (Mabtech) was added for 1 h at room temperature. After washing, streptavidin alkaline phosphatase (Mabtech) was added for 1 h at room temperature. Spots were visualized by adding 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium (Bio-Rad) for 5 to 10 min. Culture medium alone and phytohemagglutinin P (PHA-P) (10 μg/ml) served as negative and positive controls, respectively. Spots were counted on an automated CTL ELISpot assay reader. The breadth (number of reactive OLP) and total magnitude (median number of spot-forming cells [SFC] per 10⁶ PBMC for all positive responses) were recorded.

The IFN-γ secreted from CTLs against target cells was detected using ELISpot assay. Briefly, effector cells were co-cultured with target cells in 96 well plates coated with anti-IFN-γ antibody (1-D1K; Mabtech Inc., Cincinnati, OH, USA). After incubation for 20 h, a biotinylated antibody specific for IFN γ (7-B6-1; Mabtech) was added and incubated for 2 h at room temperature, followed by the addition of streptavidin-alkaline phosphatase (Mabtech) for 1 h. Nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BioRad) were added to develop spots. Color development was stopped by washing with distilled water. The resulting spots were counted using a CTL-ImmunoSpot1 analyzer (Cellular Technology Ltd., Shaker Heights, OH).

IFN-γ secretion of CTLs in response to target cells was detected using ELISpot assay. Briefly, effector cells were incubated with target cells in plates coated with anti-IFNγ antibody (1-D1K; Mabtech Inc., Cincinnati, OH). After incubation for 20 hours, biotinylated antibody specific for IFN γ (7-B6-1; Mabtech) was added for 2 hours at room temperature and then streptavidin-alkaline phosphatase (Mabtech) was added for 1 hour. To develop spots, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Biorad) were added and the color development was stopped by rinsing with distilled water. The resulting spots were counted using a CTL-ImmunoSpot^® analyzer (Cellular Technology Ltd., Shaker Heights, OH)

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).

IFN-γ secretion of CTLs in response to target cells was detected using enzyme-linked immunospot assay. Briefly, effector cells were incubated with target cells in plates coated with anti-IFNγ antibody (1-D1K; Mabtech Inc., Cincinnati, OH, USA). After incubation for 20 h, biotinylated antibody specific for IFN γ (7-B6-1; Mabtech) was added for 2 h at room temperature and then streptavidin-alkaline phosphatase (Mabtech) was added for 1 h. To develop spots, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad) were added and the color development was stopped by rinsing with distilled water. The resulting spots were counted using a CTL-ImmunoSpot analyzer (Cellular Technology Ltd., Shaker Heights, OH, USA).

Pn1-specific IgG⁺ and IgA⁺ ASCs were enumerated by ELISpot, as previously described (22 (link)), in spleen and BM 14 and 56 days after immunization. MultiScreen High protein binding immobilon-P membrane plates (Millipore Corporation, Bedford, MA) were coated with 10 µg/ml Pn1 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 incubated in four threefold dilutions starting with 1 × 10⁷ cells in 100 µL of complete RPMI 1640 per well for 5 h at 37°C, washed and incubated with ALP-goat anti mouse IgG or IgA (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 R S6 ULTIMATE using ImmunoSpot R SOFTWARE (Cellular Technology Limited (CTL) Europe, Bonn, Germany).