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

Abstract

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 Eu 3ϩ , we were able to produce the conjugate (SA) z -(b) x -PVA(BCPDA) y -Eu 3ϩ . 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 (ϳ3ϫ10 Ϫ18 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.

Multiple Fluorescent Labeling of Silica Nanoparticles with Lanthanide Chelates for Highly Sensitive Time-Resolved Immunofluorometric Assays

“…An interesting feature found is that the Eu(III) chelate shows fluorescenceenhancing effects in a multiple labeling system (8 -10 ). In such a system, a carrier molecule such as streptavidin (SA) (8 ) or a polymeric compound (11,12 ) carrying hundreds of europium chelates is used to label antibody for highly sensitive TRF immunoassays. A breakthrough to further boost the multiple-labeling strategy is the development of nanoparticles containing thousands of fluorescent lanthanide chelates for TRF immunoassays (13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Lanthanide Nanoparticules as Photoluminescent Reporters

“…The high luminescence intensity of particulate lanthanide reporters favors their application in immunoassays, where extreme sensitivity is required. The same objective has also been addressed using lanthanide labeled streptavidin macroconjugates of thyroglobulin [103], polyvinylamine [104,105], polyaminoacids [106], or precisely assembled conjugates of fluorescent proteins [107]. However, the high specific activity does not solely provide an improved sensitivity in a two-site immunoassay.…”

Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

“…In other words, the method does not allow collection of site-specific or morphological information, neither does it allow repeated and postponed expression analysis. In order to be applicable in imaging applications, and in postponed analysis, the DELFIA technique should be replaced by direct lanthanide fluorimetry (non-enhancement fluorimetry) that relies on stable fluorescent lanthanide chelates (23)(24)(25)(26). Such a stable chelate of Eu(III) was used in the present study, and these are also commercially available.…”