Use of the biotinylated antibody DAKO-ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells.
A method for the use of a biotinylated antibody (DAKO-ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC-conjugated antibody (NCL-5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating inflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio-ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. 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.
Brotherick
I
I
Department of Surgery, Medical School, University of Newcastle, Denmark.
Lennard
T W
TW
Cook
S
S
Johnstone
R
R
Angus
B
B
Winthereik
M P
MP
Shenton
B K
BK
D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D007633:Keratins / Q000032:analysis* / Q000276:immunology; D011869:Radioligand Assay; D011960:Receptors, Estrogen / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011960:Receptors, Estrogen; D007633:Keratins