title: DNA-directed assembly of antibody-fluorophore conjugates for quantitative multiparametric flow cytometry.
abstract: Multiparametric flow cytometry offers a powerful approach to single-cell analysis with broad applications in research and diagnostics. Despite advances in instrumentation, progress in methodology has lagged. Currently there is no simple and efficient method for antibody labeling or quantifying the number of antibodies bound per cell. Herein, we describe a DNA-directed assembly approach to fluorescent labeling that overcomes these barriers. Oligonucleotide-tagged antibodies and microparticles can be annealed to complementary oligonucleotides bearing fluorophores to create assay-specific labeling probes and controls, respectively. The ratio of the fluorescence intensity of labeled cells to the control particles allows direct conversion of qualitative data to quantitative units of antibody binding per cell. Importantly, a single antibody can be labeled with any fluorophore by using a simple mix-and-match labeling strategy. Thus, any antibody can provide a quantitative probe in any fluorescent channel, thus overcoming major barriers to the use of flow cytometry as a technique for systems biology and clinical diagnostics.
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, 929 E. 57th St, Chicago, IL 60637 (USA).
authors.forename: Amy C
authors.initials: AC
authors.lastname: Flor
authors.forename: Jimmy H
authors.initials: JH
authors.lastname: Williams
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Blaine
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Duggan
authors.forename: Anne I
authors.initials: AI
authors.lastname: Sperling
authors.forename: David A
authors.initials: DA
authors.lastname: Schwartz
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Kron
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000906:Antibodies; D000941:Antigens; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA
keywords: antibodies; flow cytometry; fluorescent probes; hydrazones; oligonucleotides