Brotherick I, Lennard T W, Wilkinson S E, Cook S, Angus B, Shenton B K
Department of Surgery, Medical School, University of Newcastle Upon Tyne, United Kingdom.
Cytometry. 1994 Jul 1;16(3):262-9. doi: 10.1002/cyto.990160311.
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 simple, 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.
