title: Dynamic quantification of antigen molecules with flow cytometry.
abstract: Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia.
authors: A E
authors: AE
authors: Moskalensky
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia.
authors: A V
authors: AV
authors: Chernyshev
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia.
authors: M A
authors: MA
authors: Yurkin
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia.
authors: V M
authors: VM
authors: Nekrasov
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia; JSC "Vector-Best", 630559 Koltsovo, Russia.
authors: A A
authors: AA
authors: Polshchitsin
authors: Department of Genetics, Stanford University School of Medicine, 279 Campus Drive, 94305 Stanford, CA, USA.
authors: D R
authors: DR
authors: Parks
authors: Department of Genetics, Stanford University School of Medicine, 279 Campus Drive, 94305 Stanford, CA, USA.
authors: W A
authors: WA
authors: Moore
authors: Division of Immunology and Rheumatology, Stanford University School of Medicine, 269 Campus Drive, 94305 Stanford, CA, USA.
authors: A
authors: A
authors: Filatenkov
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Novosibirsk State University, 2 Pirogova, 630090 Novosibirsk, Russia; Novosibirsk State Medical University, 52 Krasny Prospect, 630091 Novosibirsk, Russia.
authors: V P
authors: VP
authors: Maltsev
authors: Institute of Chemical Kinetics and Combustion, 3 Institutskaya, 630090 Novosibirsk, Russia; Department of Genetics, Stanford University School of Medicine, 279 Campus Drive, 94305 Stanford, CA, USA. Electronic address: orlova@stanford.edu.
authors: D Y
authors: DY
authors: Orlova
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D016827:CD8 Antigens / Q000032:analysis* / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D016827:CD8 Antigens
keywords: Antibody–antigen binding; Antigen molecules quantification; CD8α antigen concentration; Flow cytometry; Mathematical model; Reaction rate constant