The main disadvantage in ordinary agglutination immunoassay is difficulties to distinguish between specific and non-specific particle aggregation. We proposed to use Scanning Flow Cytometry to the kinetic study of the initial stages of agglutination process. The main advantage of the Scanning Flow Cytometry is a possibility to measure angular dependency of the light scattered by a single particle, an indicatrix. The most promising field for application of the indicatrix technology is a characterization of non-spherical particles. Validity of proposed method was verified by simultaneous measurements of the light scattering and fluorescence signal. We used Wentzel-Kramers-Brillouin approximation to simulate light scattering from two glued spheres and to explain the results obtained from measured indicatrices. To show an applicability of the proposed technique, the kinetic experiments were performed on latex particle covered with BSA (diameter 1.8 μm). Kinetics of dimer fraction growth initiated by mixing BSA-covered latex particles with anti-BSA immuoglobulins IgG was studied. In order to evaluate kinetic rate constant simple kinetic model involved only dimer growth reaction was applied for data treatment. Two kinetic rate constant for dimer fraction growth kB=2.88•10-12 cm3s-1 and kA=0.85•10-12 cm3s-1 were evaluated for two samples with the same origin but with different prehistory.
We have studied the kinetics of binding of dissolved antigens to antibody-receptors on the surface of the intact living cells. We have combined the Scanning Flow Cytometry with fluorescent immunoassay. Measurements of the kinetics were carried out for rabbit anti-mouse immunoglobulins that bind to receptors of the mouse hybridomas. Anti-mouse immunoglobulins were labeled with fluorescein isothiocyanate molecules (FITC). The individual cells were identified from the native Scanning Flow Cytometer records in our experiment. The kinetics of the formation of antigen-antibody complexes was measured from the fluorescence from the individual cells. A kinetic scheme for the heterogeneous reactions between ligands and receptors has been suggested. This scheme utilizes the concept of a distribution function of an amount of active receptors on the cell surface. The analytical solution for the system of the kinetic equations has been found in the reaction-limited approximation. The experimental kinetics were fitted by this mathematical model to determine the rate constant for binding reaction of the used antigen-antibody pair.
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