HTRF Human Total EGFR EX19DEL Detection Kit HTRF®

The HTRF Total-EGFR EX19DEL assay quantifies the expression level of EGFR when Exon 19 is deleted  in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-EGFR EX19DEL assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of EGFR EX19DEL in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total-EGFR EX19DEL  HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored

Detection of Total-EGFR EX19DEL with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HCC827 cells were seeded in a 96-well culture-treated plate under 30,000 cells / well in complete culture medium, and incubated overnight at 37° C, 5% CO2.The cells were then treated with increasing concentrations of PROTAC® (MS39-SJF1528-Gefitinib based PROTAC 3-MS154 and SJF1521) for 16H at 37°C, 5%CO2. After cell lysis with 50 µL of supplemented lysis buffer #4, 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total EGFR DEL19 detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, the results obtained show a dose-dependent decrease in mutant EGFR Del19  in HCC827 cells induced by the PROTAC® molecules, and DC50* were assessed for each compound. In the same experimental conditions, no cytotoxic effects were observed as evidenced by constant ATP levels measured with ATPlite^TM assay.

* DC50  corresponds to the concentration of the degrader at which 50% of the targeted protein is degraded.

H1650 cells were plated in 96-well plates (10,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for EGFR, ERBB2, ERBB3, and ERBB4 as well as with a negative control siRNA. After a 48h incubation, the cells were lysed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total EGFR DEL19 detection antibodies.

EGFR siRNA led to a 92% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed in cells transfected with ERBB2, ERBB3, or ERBB4 siRNAs. In the same experimental conditions, the silencing of the EGFR  members had no effect on cell proliferation or viability as evidenced by constant ATP levels measured with ATPlite^TM assay.

These results demonstrate that the HTRF Total EGFR DEL19 assay is specific and does not detect other HER/ErbB family members.

Total EGFR DEL19 expression levels were assessed with HTRF Total EGFR DEL19 kit in different cell lines: H1975 and H3255 cells (expressing EGFR L858R mutant), A431 & A549 cells (expressing wild type EGFR), and H1650 & HCC827 (expressing the EGFR DEL19 mutant).

The different cell lines were cultured in T175 flasks in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking. 16 µL of each lysate diluted by 2 were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total EGFR DEL19 detection reagents.

As expected, no signal was detected in the H1975, H3255, A431, and A549 cell lines, demonstrating the specificity of the HTRF Total EGFR DEL19 kit to detect mutated EGFR DEL19 only.

HCC827 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total EGFR DEl19 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total EGFR DEL19 assay is 64 times more sensitive than the Western Blot technique.

EGFR is a receptor tyrosine kinase that belongs to the ErbB family. Binding of EGFR ligands drives receptor homodimerization or hetero-dimerization, leading to the activation of the EGFR tyrosine kinase domain and specific tyrosine residues.

The phosphorylated tyrosine residues then provide docking sites for a variety of factors that induce downstream activation of several signal transduction cascades.

The signals transmitted from the EGF receptor to the nucleus lead to the regulation of various biological functions such as cell proliferation, differentiation, survival, adhesion, migration, and angiogenesis.

Mutations that lead to overexpression or hyperactivity of EGFR are associated with a number of cancers, making EGFR a key target for anti-cancer therapies.