Homogenous time-resolved fluorescence (HTRF)
HTRF® is a TR-FRET based technology from Cisbio Bioassays International. This technology uses the principles of both TRF and FRET. The HTRF® donor fluorophore is either Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate). (Lumi4™-Tb (Tb2+ cryptate) results from collaboration between Cisbio and Lumiphore Inc.)
Both donors have the long-lived emissions of lanthanides coupled with the stability of cryptate encapsulation. XL665, a modified allophycocyanin, is the HTRF® primary acceptor fluorophore. d2 represents a second generation of acceptor characterized by an organic structure 100 times smaller.
HTRF® is a highly flexible chemistry that measures biomolecular complexes of many different sizes. This includes assessment of small phosphorylated peptides, immunoassays for quantifying large glycoproteins, receptor tyrosine kinase activity using membrane preparations and indirect detection (via secondary antibodies) of tagged complexes such as CD28/CD86 binding (see figure to the right).
The following BMG LABTECH microplate readers are certified HTRF®-compatible by Cisbio:
Cellular and biochemical HTRF assays measured in 1536-well microplates
Miniaturization of an HTRF methyltransferase assay that detects histone modifying activity
The fluorescence ratio associated with HTRF® readout is a correction method developed by Cisbio and covered by the US patent 5,527,684 and its foreign equivalents. The dual simultaneous fluorescence time-resolved measurement is covered by Cisbio's US patent 6,352,672 and its foreign equivalents. Cisbio has granted BMG LABTECH a license for the promotion of these methods with its microplate readers. The application of said methods is strictly limited to the use of HTRF® reagents and technology, excluding any and all other TR-FRET technologies.