Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity

被引：11

作者：

Ferrer, M ^{[1
]}

Zuck, P

Kolodin, G

Mao, SS

Peltier, RR

Bailey, C

Gardell, SJ

Strulovici, B

Inglese, J

机构：

[1] Merck Res Labs, Dept Automated Biotechnol, N Wales, PA 19454 USA

[2] Merck Res Labs, Dept Biol Chem, West Point, PA 19486 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2003年 / 317卷 / 01期

关键词：

HTRF; CPB; HTS; miniaturization;

D O I：

10.1016/S0003-2697(03)00048-4

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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-RYRGLM VGGVVR(-OH)) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLNIVGGVV(-OH)) 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. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：94 / 98

页数：5

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