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Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.
- Source :
-
Analytical biochemistry [Anal Biochem] 2003 Jun 01; Vol. 317 (1), pp. 94-8. - Publication Year :
- 2003
-
Abstract
- 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.
- Subjects :
- Aminocaproates chemistry
Aminocaproates pharmacology
Animals
Antibodies, Monoclonal chemistry
Antibodies, Monoclonal immunology
Biotin chemistry
Carboxypeptidase B analysis
Carboxypeptidase B antagonists & inhibitors
Carboxypeptidase B chemistry
Enzyme Inhibitors pharmacology
Europium chemistry
Fluorescence Resonance Energy Transfer
Miniaturization
Oligopeptides chemistry
Oligopeptides metabolism
Phycocyanin chemistry
Sensitivity and Specificity
Streptavidin chemistry
Swine
Carboxypeptidase B metabolism
Fluorescent Antibody Technique methods
Subjects
Details
- Language :
- English
- ISSN :
- 0003-2697
- Volume :
- 317
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12729605
- Full Text :
- https://doi.org/10.1016/s0003-2697(03)00048-4