A new approach for fluorescence correlation spectroscopy (FCS) based immunoassays.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring physicochemical properties, such as concentration and diffusion constant, of bio-molecules in complex mixtures. Although, as such, FCS is well suited for development of homogeneous immunoassays, a major obstacle lies in the relatively high molecular weight of antibodies. This is because in FCS discrimination between unbound fluorescently-labelled antibodies and the same antibodies bound to immune complexes is based on the difference of their respective diffusion coefficients. To overcome this limitation we here propose to use a fluorescently-labelled tag which has two crucial properties: (a) its molecular weight is significantly lower than that of an antibody and (b) it is capable to discriminate between free antibodies and immune complexes. We have evaluated the feasibility of this approach in a model system consisting of mouse monoclonal IgG directed against the Lewis X antigen, and Protein A as a low molecular weight tag.