A microtiter-based assay for the detection of protein tyrosine kinase activity.
A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
Cleaveland
J S
JS
Bristol-Myers Squibb, Wallingford, Connecticut 06492.
Kiener
P A
PA
Hammond
D J
DJ
Schacter
B Z
BZ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000906:Antibodies / Q000276:immunology; D001322:Autoanalysis; D001667:Binding, Competitive; D002227:Carbazoles / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000502:physiology; D026121:Indole Alkaloids; D009113:Muramidase / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000276:immunology; D019311:Staurosporine; D013379:Substrate Specificity / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology
D000470:Alkaloids; D000906:Antibodies; D002227:Carbazoles; D026121:Indole Alkaloids; D010761:Phosphorus Radioisotopes; D019000:Phosphotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; C049985:staurosporine aglycone; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D010744:Phosphoric Monoester Hydrolases; D009113:Muramidase; D019311:Staurosporine