Alkaline phosphatase conjugated abs to igg

For ELISA, 96-well plates (2595; Costar) were coated with 2 μg/mL NP₃₀-BSA or 10 μg/mL OVA (Biosearch Technologies and Sigma-Aldrich) in PBS overnight. Plates were washed twice (0.5% BSA, 0.1% Tween 20 in PBS), blocked for 1 hr (0.5% BSA in PBS), and washed twice, and serially diluted samples were addedfor 1 hrat RT. Plates werewashedthree times and horseradish peroxidase (HRP)-conjugated detection Abs for IgG (Bethyl Laboratories) were added for 1 hr, washed three times, and tetramethylbenzidine substrate was added (BD Biosciences). Reaction was stopped using 2 N H₂SO₄ and read at 450 nm. Ab quantification was calculated based on NP- or OVA-specific monoclonal standards (H33lγ1 or OVA-14) and reported as relative units (RU). For ELISPOTs, Immobilon-P plates (Millipore) were coated overnight at 4°C with 2 μg/mL NP₃₀-BSA or 10 μg/mL OVA in PBS. Plates were washed twice then blocked for 2 hr. BM cells were isolated; RBCs were lysed with ACK, and then incubated for 3 hr at 37°C in RPMI. The plates were washed three times, and then incubated with alkaline phosphatase-conjugated Abs to IgG (SouthernBiotech). Plates were washed five times and developed using NBT reagent (Sigma-Aldrich).