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. 2018 Jul 16;23(9):919–929. doi: 10.1177/2472555218786165

Figure 2.

Figure 2.

(A) TR-FRET-based detection of free and total BTK. The distinct, nonoverlapping emission spectra of Tb allows it to serve as a common fluorescence donor for two spectrally distinct acceptors: G2-streptavidin (SA)-bound biotinylated tirabrutinib (to detect free BTK) and D2-coupled anti-BTK antibody that binds to a different BTK epitope (to detect total BTK). TR-FRET readout values are the ratio of acceptor to donor emissions for free BTK (520 nm/615 nm) and total BTK (665 nm/615 nm). This multiplexed assay allows detection of both free and total BTK simultaneously in the same sample. (B) Principle of the BTK occupancy assay. In the absence of tirabrutinib, 100% of BTK in the sample is unbound. The free BTK is captured by the biotinylated tirabrutinib probe and measured by the Tb-to-G2 TR-FRET readout. In the presence of a saturating dose of tirabrutinib, all BTK in the sample is drug bound. The lack of TR-FRET signal for free BTK indicates 100% BTK occupancy.