Journal of Biotechnology 107 (2004) 185–192 A new approach for fluorescence correlation spectroscopy (FCS) based immunoassays Oleg A. Mayboroda a,∗ , Alexandra van Remoortere a , Hans J. Tanke b , Cornelis H. Hokke a , André M. Deelder a a Department of Parasitology, Leiden University Medical Centre (LUMC), L4-Q, P.O. Box 9600, 2300RC Leiden, The Netherlands b Department of Molecular Cell Biology, Leiden University Medical Centre (LUMC), Leiden, The Netherlands Received 30 January 2003; received in revised form 24 September 2003; accepted 9 October 2003 Abstract 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 devel- opment 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. © 2003 Elsevier B.V. All rights reserved. Keywords: Fluorescence correlation spectroscopy; Protein A; Lewis X; Immunoassay 1. Introduction Analysis of body fluids or tissue extracts should ideally combine minimal sample consumption with high sensitivity and specificity. Fluorescence correla- tion spectroscopy (FCS) is one of the few methods, which combines these characteristics. In FCS the fluctuations δF(t) of an average fluores- cence signal 〈F〉 are measured in a small (∼0.25 fl) illuminated volume and the autocorrelation function ∗ Corresponding author. Fax: +31-71-526-6907. E-mail address: o.a.mayboroda@lumc.nl (O.A. Mayboroda). (ACF) of the fluorescence intensity G(τ ) is used to obtain information about the molecular processes re- sponsible for these fluctuations (Elson and Magde, 1974; Thompson, 1991). Analysis of the shape and magnitude of ACF by applying a mathematical model provides the average number of fluorescent molecules per measuring volume and their characteristic dif- fusion time. These parameters can then be used to calculate the diffusion coefficient, as well as the size and the concentration of the molecules. In terms of biochemical applications, the method can be used for studying, e.g. molecular aggregation in solution and receptor–ligand interactions, and for the detection 0168-1656/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2003.10.007