Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio‐ligand binding assay

Abstract

A method for the purification, conjugation, and use of EGF-R antibody to quantify EGF receptors on tumour cells by flow cytometry is described. The quantification of both internal and external EGF receptors was determined by treatment with saponin, rendering the cells permeable to the EGF-R antibody. Using QCS bead standards, the number of EGF-R binding sites per cell was assessed. Results were compared with conventional EGF-R quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for EGF-R quantification described is sim-ple, rapid, and reproducible. The assay may be of particular value in measuring EGF-R on urgent clinical samples or those that are too small (such as breast aspirates) for measurement by the radio-ligand binding assay. Advantages of this assay over the radio-ligand binding assay include reduction in use of radio-labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. In addition, flow cytometry offers the possibility of selecting cell phenotypes by gating as well as live/dead cells by using multi-parameter flow cytometry. 0 1994 Wiley-Liss, Inc.

Gefitinib (‘Iressa’, ZD1839) inhibits the growth response of bladder tumour cell lines to epidermal growth factor and induces TIMP2

“…These cell lines differ in their EGFR numbers, with RT4 and RT112 cells containing 1.9 Â 10 4 and 9 Â 10 3 receptors per cell, respectively, as measured by FACS analysis (Brotherick et al, 1994). All cells were negative for mycoplasma.…”

Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study

“…The method described by Brotherick et al (1994Brotherick et al ( , 1995a was used. Incubation of Quantum Simply Cellular beads (QSC, Flow Cytometry Standards Corp., NC, USA) with an excess of ER-and PR-conjugated antibody was performed to saturate all binding sites.…”

“…Use of this technique has been developed for the analysis of breast cancer where it has shown excellent correlation with the radioligand binding assay (Brotherick et al, 1994).…”

Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study

“…The method described by Brotherick et al (1994 was used. Incubation of Quantum Simply Cellular beads (QSC, Flow Cytometry Standards Corp., NC, USA) with an excess of ER-and PR-conjugated antibody was performed to saturate all binding sites.…”

“…A cut-off value of 3000 sites per cell was used, with numbers below this regarded as negative for the receptors. This was determined using earlier published work comparing the flow cytometric assay with radio-ligand binding assays in breast carcinoma (Brotherick et al, 1994).…”