Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.
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-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]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.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. marc_ferreralegre@merck.com
Marc
M
Ferrer
Paul
P
Zuck
Garrett
G
Kolodin
Shi Shan
SS
Mao
Richard R
RR
Peltier
Carolyn
C
Bailey
Stephen J
SJ
Gardell
Berta
B
Strulovici
James
J
Inglese
D000614:Aminocaproates / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D043424:Carboxypeptidase B / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005063:Europium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique / Q000379:methods*; D008904:Miniaturization; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010798:Phycocyanin / Q000737:chemistry; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry; D013552:Swine
D000614:Aminocaproates; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C002497:allophycocyanin; D010798:Phycocyanin; D005063:Europium; D001710:Biotin; D019809:Streptavidin; D043424:Carboxypeptidase B