Development of an HTRF assay using a biotinylated substrate recognized by a specific anti-CBP antibody labeled with EuK
HTRF toolbox for an enzymatic assay in 1536 with Z’ of 0.8. Using anti-CBP sbubstrate antibody custom labeling with EuK (by Merck).
An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu3+ -cryptate ([Eu3+ ]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z’ >0.5) assay for high-throughput screening.
Anal Biochem. 2003;317(1):94-8.