An immunoassay for assessment of receptor tyrosine kinase autophosphorylation

We have developed an immunoassay that can be used to assess autophosphorylation activity of receptor tyrosine kinases, yet does not require the use of synthetic peptide substrates or anti-receptor antibodies. In the assay described here, receptor autophosphorylation and detection take place entirely within the wells of 96 well microplates coated with Protein G and an anti-phosphotyrosine antibody. As the kinase reaction takes place, the antibodies capture the phosphorylated products in situ. Phosphorylation levels of captured receptors are measured by scintillation counting. Assay parameters were validated using A431 cell extracts containing EGF Receptor. The autophosphorylation capture assay is a simple and rapid method which can be adapted for use with robotics for qualitative HTS of potential inhibitors to any tyrosine kinase of interest.