Visualization of an immunological reaction between single antigen and antibody molecules by optical chromatography

Optical chromatography, which is a technique for the separation of microscopic particles using a radiation force, is applied to kinetic studies of an immunoreaction. Polystyrene beads coated with antibodies react with antigens to produce conjugated beads, which comprise two beads and some antigens. The resulting conjugated beads are separated from the single beads. The detection limit of the antigen is <1 ng ml^-1, which is smaller than that reported in a previous paper [T. Hatano, T. Kaneta, T. Imasaka, Anal. Chem. 69 (1997) 2711], and can be achieved using 2 μm-diameter beads. The sensitivity in the present method can be further enhanced by increasing the particle size. In addition, the dissociation reaction of the conjugated beads is evaluated by using an on-column reaction technique, which allows the direct monitoring of the formation and dissociation of the conjugated beads. The rate constant for the dissociation of the conjugated beads is determined to be 4.0 x 10^-3 s^-1 for the case of conjugated beads combined by a single bond.