Standardization of an enzyme-linked immunosorbent assay for the determination of protein tyrosine kinase activity.
A procedure for an enzyme-linked immunosorbent assay for the determination of protein tyrosine kinase (PTK) activity from cytosolic and solubilized membrane fractions from breast cancers, is described. The general PTK substrate poly(GluNa, Tyr) 4:1 is coated to the wells of a microtiter plate. After incubation with PTK sample and ATP the amount of phosphorylated tyrosyl residues is quantitated with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay is optimized with respect to coating and phosphorylation conditions. The signal is linear with phosphorylation time and with sample protein concentrations in a sufficiently wide range. The assay is standardized by using both internal and external standards. A lyophilized rat spleen extract is used as an external standard. Its PTK activity, determined by established quantitative methods, can be used to calculate the activity of the breast cancer samples. To eliminate day-to-day variations an internal standard, consisting of BSA-coupled phosphotyrosine, is coated to some wells of the microtiter plate. Interassay variation can be minimized by determination of the ratio of optical densities from internal and external controls. Its variation appeared to be less than 18%. Intraassay variation appears to be < 6%. PTK activities measured with this assay correlated well with those of a nonradioactive dot-blot assay and with conventional radioactive assays in which [32P]ATP is used as the substrate. Compared to these assays it appeared to be more sensitive and far more easy to perform.
Schraag
B
B
Department of Hematology, University Hospital Utrecht, The Netherlands.
Staal
G E
GE
Adriaansen-Slot
S S
SS
Salden
M
M
Rijksen
G
G
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology; D002623:Chemistry Techniques, Analytical / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005260:Female; D006801:Humans; D008297:Male; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013154:Spleen / Q000737:chemistry; D014020:Tissue Extracts / Q000032:analysis; D014238:Trichloroacetic Acid; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D003902:Detergents; D014020:Tissue Extracts; D019000:Phosphotyrosine; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D014238:Trichloroacetic Acid; D011505:Protein-Tyrosine Kinases