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

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.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling.

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called "apoptotic ring"). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.