title: Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by flow cytometry.
abstract: Quantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13-6 (Kindler-Röhrborn et al.: Differentiation 30:53-60, 1985). For these non-disjunct distributions of fluorescence intensities, the cut-off border between antigen-positive and antigen-negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen-negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13-6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard-plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage.
authors.affiliation: Institute of Cell Biology (Cancer Research), West German Cancer Center Essen.
authors.forename: R
authors.initials: R
authors.lastname: Dux
authors.forename: A
authors.initials: A
authors.lastname: Kindler-Röhrborn
authors.forename: K
authors.initials: K
authors.lastname: Lennartz
authors.forename: M F
authors.initials: MF
authors.lastname: Rajewsky
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology*; D001921:Brain / Q000196:embryology; D001932:Brain Neoplasms / Q000276:immunology / Q000473:pathology* / Q000648:ultrastructure; D002138:Calibration; D002460:Cell Line; D000939:Epitopes; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D009374:Neoplasms, Experimental / Q000276:immunology / Q000473:pathology* / Q000648:ultrastructure; D011863:Radioimmunoassay; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D000939:Epitopes