Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes

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Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes. / Khalo, Irina V.; Kozyreva, Viktoriya S.; Vakhrushev, Roman V. et al.

In: Cytometry Part A, Vol. 93, No. 7, 01.07.2018, p. 695-705.

Research output: Contribution to journal › Article › peer-review

BibTeX

@article{edc96561fd1643388fc92f1f4631ed43,

title = "Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes",

abstract = "We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand–receptor (antigen–antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct (k+ (5.6 ± 0.2) × 107 M−1 min−1) and reverse (k- (1.3 ± 0.2) × 10−2 min−1) rate constants of ligand–receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.",

keywords = "calibration-free flow cytometry, CD14 receptors, dynamic quantitative immunoassay, human blood leukocytes, ligand–receptor binding kinetics, mathematical model, ligand-receptor binding kinetics, STANDARDS, INDIRECT IMMUNOFLUORESCENCE, DESCRIPTORS, MASS CYTOMETRY, KINETICS, MONOCYTE SUBSETS, MONOCLONAL-ANTIBODIES, CELL, BLOOD, Immunoassay/methods, Humans, Flow Cytometry/methods, Leukocytes/chemistry, Immunoglobulin G/chemistry, Protein Binding, Binding Sites, Antibody, Lipopolysaccharide Receptors/chemistry",

author = "Khalo, {Irina V.} and Kozyreva, {Viktoriya S.} and Vakhrushev, {Roman V.} and Patlai, {Daria S.} and Shilova, {Anna N.} and Karpenko, {Andrei A.} and Yurkin, {Maxim A.} and Moskalensky, {Alexander E.} and Strokotov, {Dmitry I.} and Maltsev, {Valeri P.} and Chernyshev, {Andrei V.}",

note = "{\textcopyright} 2018 International Society for Advancement of Cytometry.",

year = "2018",

month = jul,

day = "1",

doi = "10.1002/cyto.a.23494",

language = "English",

volume = "93",

pages = "695--705",

journal = "Cytometry. Part A : the journal of the International Society for Analytical Cytology",

issn = "1552-4922",

publisher = "Wiley-Liss Inc.",

number = "7",

}

RIS

TY - JOUR

T1 - Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes

AU - Khalo, Irina V.

AU - Kozyreva, Viktoriya S.

AU - Vakhrushev, Roman V.

AU - Patlai, Daria S.

AU - Shilova, Anna N.

AU - Karpenko, Andrei A.

AU - Yurkin, Maxim A.

AU - Moskalensky, Alexander E.

AU - Strokotov, Dmitry I.

AU - Maltsev, Valeri P.

AU - Chernyshev, Andrei V.

PY - 2018/7/1

Y1 - 2018/7/1

N2 - We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand–receptor (antigen–antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct (k+ (5.6 ± 0.2) × 107 M−1 min−1) and reverse (k- (1.3 ± 0.2) × 10−2 min−1) rate constants of ligand–receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

AB - We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand–receptor (antigen–antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct (k+ (5.6 ± 0.2) × 107 M−1 min−1) and reverse (k- (1.3 ± 0.2) × 10−2 min−1) rate constants of ligand–receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

KW - calibration-free flow cytometry

KW - CD14 receptors

KW - dynamic quantitative immunoassay

KW - human blood leukocytes

KW - ligand–receptor binding kinetics

KW - mathematical model

KW - ligand-receptor binding kinetics

KW - STANDARDS

KW - INDIRECT IMMUNOFLUORESCENCE

KW - DESCRIPTORS

KW - MASS CYTOMETRY

KW - KINETICS

KW - MONOCYTE SUBSETS

KW - MONOCLONAL-ANTIBODIES

KW - CELL

KW - BLOOD

KW - Immunoassay/methods

KW - Humans

KW - Flow Cytometry/methods

KW - Leukocytes/chemistry

KW - Immunoglobulin G/chemistry

KW - Protein Binding

KW - Binding Sites, Antibody

KW - Lipopolysaccharide Receptors/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85052678810&partnerID=8YFLogxK

U2 - 10.1002/cyto.a.23494

DO - 10.1002/cyto.a.23494

M3 - Article

C2 - 30110130

AN - SCOPUS:85052678810

VL - 93

SP - 695

EP - 705

JO - Cytometry. Part A : the journal of the International Society for Analytical Cytology

JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology

SN - 1552-4922

IS - 7

ER -

ID: 16330276