Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor.
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(50) 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.
Angeles
T S
TS
Department of Biochemistry, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, USA.
Lippy
J S
JS
Yang
S X
SX
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D051379:Mice; D020932:Nerve Growth Factor / Q000378:metabolism; D010766:Phosphorylation; D020917:Receptor, trkA / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
D000906:Antibodies; D020932:Nerve Growth Factor; D020917:Receptor, trkA