Figure 3.

(A) Assay range of quantitation. A set of six experiments (in quadruplicate) using full-length purified rBTK protein was used to generate independent standard curves for free and total BTK. The lower limit of quantitation of the assay was 12 ng/mL, and the upper limit was 166 ng/mL. The BTK concentration in cell samples in subsequent experiments was interpolated from these standard curves. (B) Assay accuracy and precision. Low percent CV (solid lines) in interassay performance demonstrated precision of the assay, and percent recovery (dotted lines) after spikes of 110, 8, and 0 nM tirabrutinib demonstrated accuracy of the assay. Data represent mean values from four independent experiments in quadruplicate. (C) Specificity of the assay for BTK inhibition was demonstrated by detection of free and total BTK in titrated lysates of Ramos B cells (light purple dots) but not Jurkat T cells (pink dots). BTK expression was lower in PBMCs (dark purple dots) than in Ramos cells. The decrease in TR-FRET at high Ramos cell concentration reflects analyte saturation. Representative data are shown from three or more experiments.