Joint application of an ARC-probe and antibody in homogeneous TR-FRET assay for determination of the concentration of protein kinase Pim2



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Förster resonance energy transfer (FRET) is a routinely apply photoluminescence technology in high throughput screening in drug discovery. It involves the transfer of energy between two luminescence molecules, the donor and the acceptor in close proximity. In this thesis, we describe the application of the approach exploiting time-resolved measurement of intensity of FRET (TR-FRET) from terbium donor luminophore to fluorescent dye Alexa Fluor 647, for the measurement of the concentration of a specific protein kinase Pim2 in a solution-phase homogenous assay. This method is based on the simultaneous binding of a specific PK to both a luminophore ARC-type probe inhibitor and a fluorescently labelled monoclonal antibody specific to the PK. In case specific IgG labelled with Alexa Fluor 647 acting as acceptor fluorophore is combined in solution with donor luminophore (lanthanide chelate labelled) ARC-probe and the protein kinase of interest, the formation of a triple complex antibody:protein kinase:ARC-probe can be measured by time-resolved measurement of FRET intensity. First, we were able to determine the active concentration of the PK, using a flurescently labelled ARC-type inhibitor of the PK’s active site. Second, we were able to prove the detection of a specific PK by formation of high a FRET signal between a luminophore ARC-type probe PK inhibitor as donor and a fluorescently labelled antibody specific to the PK as acceptor. In addition, formation of the triple complex between ARC-type inhibitor:PK:mAb(Pim2) was confirmed by addition of another ARC-type inhibitor in the well were maximal FRET signal ratio was observed. The determined limit of quantification of the assay was 8 nM, that points to high sensitivity of this homogeneous analytical method. The approach described in this thesis, could be used as a rapid medical diagnostic tool for PK detection and analysis.