Standardization of an enzyme-linked immunosorbent assay for the determination of protein tyrosine kinase activity.

摘要: Abstract A procedure for an enzyme-linked immunosorbent assay the determination of protein tyrosine kinase (PTK) activity from cytosolic and solubilized membrane fractions breast cancers, is described. The general PTK substrate poly(GluNa, Tyr) 4:1 coated to wells a microtiter plate. After incubation with sample ATP amount phosphorylated tyrosyl residues quantitated phosphotyrosine specific antibodies secondary peroxidase-labeled antibody. optimized respect coating phosphorylation conditions. signal linear time concentrations in sufficiently wide range. standardized by using both internal external standards. lyophilized rat spleen extract used as standard. Its activity, determined established quantitative methods, can be calculate cancer samples. To eliminate day-to-day variations standard, consisting BSA-coupled phosphotyrosine, some Interassay variation minimized ratio optical densities controls. appeared less than 18%. Intraassay appears