Alkaline phosphatase substrate solution

NP-specific ASCs in spleen or ASCs in the iPC differentiation system were detected using an ELISpot assay on a MultiScreen 96-well filtration plate (Millipore, MSIPS4W10) coated with 10 μg/ml NP₂₉-BSA or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml). Serially diluted cells were added to individual wells in triplicate and incubated for 6 h at 37 °C with 5% CO₂. Anti-NP IgG1, Anti-IgG1 or Anti-IgE spots were revealed by biotin-conjugated anti-mouse IgG1 or IgE Ab (Southern Biotechnology, 1070-08) in conjunction with Av-HRP and AEC substrate (Vector Laboratories, A-2004& SK-4200).
For ELISA, flat-bottom 96-well plates (NUNC, 439454) were coated with NP-BSA (10 µg/ml) or anti-mouse kappa-UNLB and anti-mouse lambda-UNLB (2.5 µg/ml) in PBS. Nonspecific binding was blocked with 0.5% BSA in PBS. Serum samples were serially diluted in 0.5% BSA in PBS and were incubated in blocked plates at room temperature for 2 h. Plates were incubated with biotin-conjugated anti-Ig (Southern Biotech) for 2 h and with streptavidin–alkaline phosphatase for 1 h (Roche, 1089161), and then incubated with alkaline phosphatase substrate solution containing 4-nitro-phenyl phosphate (Sigma-Aldrich, N2765) for color development, followed by quantification on Molecular Devices (VERSA max).