Alphascreen igg detection kit

A total of 5 × 10⁵ Ramos cells/well were plated in 96-well PDL-coated plates in DMEM with high glucose (GIBCO) supplemented with 10% FBS. Serial dilutions of LY3439943 in DMSO (final DMSO concentration, 0.4%) were incubated with the cells for 1 h at 37 °C. An equal volume of a covalent biotinylated BTK probe was added (final concentration, 167 nM), and the cells were incubated for another 1 h at 37 °C. The cells were lysed, and the amount of BTK was determined using the AlphaScreen IgG Detection Kit (Perkin Elmer 6760617R) and an anti-BTK antibody (Thermo Scientific Cat# MA5-14929) following the manufacturer’s instructions. The signal was measured at 615 nm with an Envision plate reader (Perkin Elmer). The absolute IC₅₀ was calculated as percent inhibition of the signal window as defined by the maximum (assay medium with 0.4% DMSO) and minimum (1 µM ibrutinib, a known BTK inhibitor) using a 4-parameter nonlinear logistic equation (GeneData Screener 13.0.5 or GraphPad Prism 7.03).

The AlphaScreen assay was performed using 384-well ProxiPlates (PerkinElmer, Boston, MA, USA). Biotinylated virus proteins or GST (negative control) were incubated with a 40-fold dilution of MAbs (hybridoma supernatant) in 15 μl of binding mixture containing reaction buffer (100 mM Tris-HCl, pH 7.5, 1 mg/ml BSA, 0.01% Tween-20) at 26°C for 30 min. Then, combine 10 μl of the detection mixture containing 0.1 μl protein G-conjugated acceptor beads and 0.1 μl streptavidin-coated donor beads (AlphaScreen IgG detection kit, PerkinElmer) in reaction buffer were incubated at 26°C for 1 h. Antigen-antibody interactions were analyzed using an Envision microplate reader (PerkinElmer).

The AlphaScreen assay was performed using 384-well Proxi Plates (PerkinElmer, Boston, MA, USA). Each biotinylated peptide or DHFR (negative control) was mixed in 15 μL of assay buffer [100 mM Tris-HCl (pH 8.0), 0.1% BSA, 0.01% Tween-20] and incubated at 26 °C for 30 min. Next, 10 μL of a detection mixture containing 0.04 μL protein G-conjugated acceptor beads and 0.04 μL streptavidin-coated donor beads (AlphaScreen IgG detection kit, PerkinElmer) were incubated in reaction buffer at 26 °C for 30 min. Antigen-antibody interactions were analyzed using an Envision microplate reader (PerkinElmer).