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


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(3+), we were able to produce the conjugate (SA)(z)-(b)(x)-PVA(BCPDA)(y)-Eu(3+). 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 specific antigen (PSA). Detection limits achieved were around 1 to 2 ng/L ( approximately 3 x 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.
Scorilas
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A
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Diamandis
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EP
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005063:Europium / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D015200:Fluoroimmunoassay / Q000379:methods / Q000592:standards; D005470:Fluorometry / Q000379:methods; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry; D051379:Mice; D010618:Phenanthrolines / Q000737:chemistry / Q000378:metabolism; D011145:Polyvinyls / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010618:Phenanthrolines; D011145:Polyvinyls; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; C008996:poly(vinylamine); D005063:Europium; D001710:Biotin; D019809:Streptavidin