Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Kevin Schooler, H. S. Wiley

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme- linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8 to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.

Original languageEnglish (US)
Pages (from-to)135-142
Number of pages8
JournalAnalytical Biochemistry
Volume277
Issue number1
DOIs
StatePublished - Jan 1 2000
Externally publishedYes

Fingerprint

Epidermal Growth Factor Receptor
Protein-Tyrosine Kinases
Assays
Chemical activation
Immunosorbents
Enzyme-Linked Immunosorbent Assay
Phosphotyrosine
Enzymes
Tyrosine
Western Blotting
Phosphorylation
ErbB Receptors
Proteins
Down-Regulation
Monoclonal Antibodies
Ligands
Antibodies

Keywords

  • Enzyme-linked immunosorbent assay (ELISA)
  • Epidermal growth factor receptor (EGFR)
  • Ratiometric
  • Tyrosine kinase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation. / Schooler, Kevin; Wiley, H. S.

In: Analytical Biochemistry, Vol. 277, No. 1, 01.01.2000, p. 135-142.

Research output: Contribution to journalArticle

Schooler, Kevin ; Wiley, H. S. / Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation. In: Analytical Biochemistry. 2000 ; Vol. 277, No. 1. pp. 135-142.
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