Measuring Anti-ING1 Antibody Levels with AlphaLISA

We performed amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using 384-well microtiter plates (white opaque OptiPlate, PerkinElmer, Waltham, MA) containing 2.5 μL 1/100-diluted sera and 2.5 μL GST-ING1 or control GST proteins (10 µg/mL), or biotinylated peptides (400 ng/mL) in AlphaLISA buffer containing 25 mM HEPES (pH 7.4), 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500, and 0.05% Proclin-300. We incubated the reaction mixtures at room temperature for 6 to 8 h, and then incubated them at room temperature in the dark for 7 to 21 days with anti-human IgG-conjugated acceptor beads (2.5 µL at 40 µg/mL) and glutathione-conjugated or streptavidin-conjugated donor beads (2.5 µL at 40 µg/mL). After many AlphaLISA trials, we concluded that an incubation time of 7 to 21 days was most suitable to obtain specific, stable, low background, and reproducible results. We read the Alpha photon counts in microtiter plates using an EnSpire Alpha microplate reader (PerkinElmer) as described previously [8 (link), 9 (link), 12 (link)–14 (link)]. Specific anti-ING1 antibody levels were obtained by subtracting the Alpha counts for the GST and buffer controls from those for the GST-ING1 protein and ING1 peptide, respectively.