Polyvinylamine-streptavidin complexes labeled with a europium chelator: a universal detection reagent for solid-phase time resolved fluorometric applications

Objectives: To describe the synthesis and characteristics of a new streptavidin-based universal detection reagent which is multiply labeled with the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic-acid (BCPDA). Methods and results: Polyvinylamine (PVA) was first labeled with biotin(b) and then with BCPDA to create (b)x-PVA-(BCPDA)y. By mixing controlled amounts of this complex with streptavidin (SA) and a fixed amount of Eu3+, we were able to produce the conjugate (SA)z-(b)x-PVA(BCPDA)y-Eu3+. This conjugate is reactive, highly fluorescent, and stable for at least 12 months. It was used to develop model solid-phase time-resolved fluoroimmunoassays for biotinylated mouse IgG and prostate spesific antigen (PSA). Detection limits achieved were around 1 to 2 ng/L ( 3×10−18 moles/assay). Conclusions: A new universal detection reagent was synthesized, which can be used in combination with biotinylated reagents (e.g., antibodies, DNA probes, etc.) for the development of highly sensitive solid-phase time-resolved fluorescence-based assays.