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Updated: Aug 17, 2026
Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays
Published on: September 19, 2018
Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors
Edit Varkondi1, Eszter Schäfer, Györgyi Bökönyi
1Rational Drug Design Laboratory CRC, Semmelweis University, Budapest, Hungary.
Abstract:
Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.
Insights
Assay variations in screening receptor tyrosine kinase inhibitors, like epidermal growth factor receptor (EGFR) tyrosine kinase (TK), stem from enzyme purity, not source. Standardizing detection methods is crucial for reliable PTK inhibitor data comparison.
Area of Science:
- Biochemistry
- Pharmacology
- Cancer Research
Background:
- Receptor tyrosine kinases (PTKs) are crucial in diseases like cancer, driving PTK inhibitor development.
- Current PTK inhibitor screening data lack comparability due to diverse assay technologies.
Purpose of the Study:
- To compare ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compounds.
- To investigate interassay variations influenced by different EGFR-TK enzyme sources and purities.
Main Methods:
- Utilized three protocols for EGFR-TK enzyme: A431 cell lysate (unpurified), affinity-purified A431 EGFR-TK, and recombinant EGFR-TK (Baculovirus Sf9 cells).
- Employed a synthetic peptide substrate and ELISA to detect phosphorylated tyrosine residues using a monoclonal antibody.
Main Results:
- Significant differences observed in enzyme activity and inhibitory effects of PD153035 across protocols.
- Unpurified A431 cell lysate showed marked variation compared to affinity-purified enzymes.
- Affinity-purified EGFR-TKs from A431 cells and recombinant sources yielded comparable results.
Conclusions:
- Enzyme purity, not the source (cell lysate vs. recombinant), is the primary driver of interassay variation in EGFR-TK inhibitor screening.
- Standardizing detection methods and normalizing EGFR activity are essential for reproducible and comparable screening data.
