Dendrimer based non-competitive fluoroimmunoassay for analysis of cortisol

Abstract

The use of immunoassays in clinical diagnostics has stimulated the development of many sensitive and highly specific techniques to measure the presence of specific antigens in samples. This research proposes a non-competitive fluoroimmunoassay method for analysis of cortisol based on the blocking of unbound sites of the capture antibody by means of a reagent that consists of a dendrimer molecule bound to cortisol. Dendrimers are ordered branched polymers. The dendrimer–cortisol complex binds to more than one antibody at the same time, so it is more strongly bound than the analyte to the immobilized antibody. In this way, when the fluorescent-labeled antigen is added, competition for the antibody occupancy allows the removal of the analyte molecules, but not the dendrimer molecule. The measured signal is directly related to the analyte concentration (i.e. cortisol). Size exclusion chromatography (SEC) shows the presence of a high molecular weight component after dendrimer–cortisol synthesis. Results show cortisol covalently attached to PAMAM (Polyamidoamine) dendrimers can be used successfully as a blocking reagent in a non-competitive assay. The non-competitive assay standard curve shows increased fluorescence as the concentration of cortisol increases, which demonstrates that cortisol is being replaced by FITC-cortisol.