Fluorescence immunoassay based on long time correlations of number fluctuations

We report the development of a fluorescence-based immunoassay technique relying on the physical phenomena of random number fluctuations and diffusion, which we review. By determining the autocorrelation of the fluctuations in the fluorescent intensity, this methid is able to measure the amount of labeled antigen or antibody that is bound to micrometer-sized carrier particles in solution. The principal advantage of this technique is its insensitivity to small, fast-diffusing sources. It also discriminates against weakly fluorescent contaminants of size comparable to the carrier particles. We demonstrate these attributes by using two model systems: a human IgG assay and an idealized system consisting of polystyrene fluorescent spheres and rhodamine dye.