Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor

被引：14

作者：

Angeles, TS ^{[1
]}

Lippy, JS ^{[1
]}

Yang, SX ^{[1
]}

机构：

[1] Cephalon Inc, Dept Biochem, W Chester, PA 19380 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2000年 / 278卷 / 02期

关键词：

D O I：

10.1006/abio.1999.4441

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Two quantitative, high-throughput cell-based assays for evaluating inhibitors of NGF-stimulated trkA phosphorylation in trkA-transfected NIH3T3 cells have been established. Both assays involve capture of the trkA receptor from cell lysates in microtiter plates coated with an anti-trk antibody. The amount of trkA phosphorylation is then measured using either an anti-phosphotyrosine antibody with a colorimetric readout or a lanthanide (europium)labeled anti-phosphotyrosine antibody with a fluorometric detection. The two assay formats exhibited at least a fivefold increase in phosphorylated trkA signal in trkA-transfected cells compared to vector control. Inhibition plots generated for trkA kinase inhibitors using the two detection systems yielded comparable IC,, values. Overall, the two assays represent a marked improvement over the standard gel-based/western blot method in terms of throughput, quantitation, and amenability to automation. (C) 2000 Academic Press.

引用

页码：93 / 98

页数：6

共 25 条

[1] Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity [J].