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 IC50 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.