A microtiter-based assay for the detection of protein tyrosine kinase activity

Journal Article · Thu Nov 01 04:00:00 EST 1990 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(90)90188-F· OSTI ID:5962728

Cleaveland, J S; Kiener, P A; Hammond, D J; Schacter, B Z ^[1]

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1. Bristol-Myers Squibb, Wallingford, CT (USA)

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 ({sup 32}P)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.

• OSTI ID: 5962728
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 190:2; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

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550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENZYME ACTIVITY
ENZYME IMMUNOASSAY
ENZYME INHIBITORS
ENZYMES
HYDROXY ACIDS
IMMUNOASSAY
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
RADIOISOTOPES
TRACER TECHNIQUES
TRANSFERASES
TYROSINE