High throughput assay for inhibitors of the epidermal growth factor receptor-associated tyrosine kinase.

We have developed a colorimetric assay for the examination of inhibitors of epidermal growth factor (EGF) receptor-associated tyrosine kinase in intact cells. EGF receptor from cells treated with inhibitors is captured by an anti-EGF receptor antibody and the phosphotyrosine content is measured by an anti-phosphotyrosine antibody. The quantitative assay does not use radioactive substances and is configured for a high-throughput format. Since it is performed in intact cells, substances lower the phosphotyrosine content on the receptor by different mechanisms will be identified. One distinct feature of the assay is that it uses the natural substrate inside the cell as compared to others using artificial substrates in an unphysiological environment. This assay is easy to perform, is reproducible, and is compatible with many organic solvents and tissue culture media. Thus, it is useful for the discovery of EGF receptor kinase inhibitors from natural products or synthetic compounds and is particularly suitable for large-scale screening.