A new assay design for clinical diagnostics based on alternative recognition elements

Herein, we present a new sandwich assay design containing a high affinity polypeptide scaffold as immobilized capture element and an antibody for detection. These polypeptide scaffolds provide a good affinity towards one antigen and can be linked to biosensor surfaces without affecting their binding capabilities. Furthermore, the small peptides are very stable, which allows for regenerating the surface several hundreds of times and thus for reuse of the biosensor. Moreover, these receptors can be synthesized with different affinities towards one antigen, which has been proven by characterizing them using a label-free detection method RIfS (reflectometric interference spectroscopy) for collecting kinetic data. Polypeptide scaffolds with different affinities have been chosen and characterized. Upon these results, sandwich-type assays have been set-up using a fluorescently labelled antibody as detection element. Thereby could be shown, that the working range of the assay can be shifted according to the affinity of the used capturing polypeptide scaffold. The scaffolds with a higher affinity towards the antigen can detect lower concentration, and in contrary, scaffolds with lower affinities can detect higher concentrations. In consequence, using this new sandwich-type assay, we avoid the complex procedure to immobilize antibodies in correct orientation, but simultaneously keep this well-known recognition element in the assay for detection. Furthermore, in addition to all the acknowledged properties of immunoassays, we add the possibility of tuning the working range of assays in distinct manner according to request. (C) 2010 Elsevier B.V. All rights reserved.