A novel and robust homogeneous fluorescence-based assay using nanoparticles for pharmaceutical screening and diagnostics

We have established a new type of homogeneous immunoassay based on nanopart icles (nanoparticle immunoassay, or NPIA) being analyzed using fluorescence intensity distribution analysis (FIDA), This method allows the characteriz ation of single fluorescently labeled molecules or particles with respect t o their molecular brightness and concentration, Upon binding of conjugates to molecules coupled to the nanoparticle surface, the brightness of the com plex scales with the number of bound conjugates, The complexes can then be distinguished accurately from free conjugate and concentrations of free and bound molecules can be determined reliably. In this study we present vario us examples of NPIAs where capture antibodies were linked to the nanopartic les, which were either artificial beads or bacteria. Two assay formats have been developed; first, direct labeling of the conjugate was used to quanti tate free antigen through competition experiments, and second, an antigen-d irected antibody was labeled to establish an assay similar to a sandwich EL ISA setup. The major advantages of a NPIA are the robustness and high signa l-to-noise ratio at short measurement times, as demonstrated with a miniatu rized experiment in a Nanocarrier(TM) holding a volume of 1 mu l/well. in a ddition to the good data quality, NPIAs are straightforward to perform beca use they require no washing steps. NPIAs open new dimensions for high throu ghput pharmaceutical screening and diagnostics. Assay development times can be reduced significantly because of a simple toolbox principle that is app licable to most types of assays.