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Preliminary examination of time‐resolved fluorometry for protein array applications
Author(s) -
Luo LiuYing,
Diamandis Eleftherios P.
Publication year - 2000
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/1522-7243(200011/12)15:6<409::aid-bio628>3.0.co;2-6
Subject(s) - biotinylation , analyte , streptavidin , chemistry , chromatography , fluorescence spectroscopy , fluorescence , protein microarray , reagent , polystyrene , fluorophore , biotin , analytical chemistry (journal) , biochemistry , microarray , physics , gene expression , organic chemistry , quantum mechanics , gene , polymer
The advantages of time‐resolved fluorometry over conventional fluorometric analysis are well known. However, time‐resolved fluorescence has not as yet found wide applications in protein microarray or other multiparametric methods of analysis. Here we describe a general method which is suitable for multiparametric and microarray analysis, based on time‐resolved fluorometry. A polystyrene surface is coated with different monoclonal antibodies, specific for certain analytes. The analyte mixtures are then universally biotinylated, using an active biotin ester. After removing excess biotin, the biotinylated samples are applied on the polystyrene surface, incubated and the excess is washed away. The bound moieties are then quantified by adding a universal detection reagent containing streptavidin, labelled with a fluorescent europium chelate. After washing and drying of the solid surface, the immobilized moieties are detected by using solid‐phase, laser‐excited time‐resolved fluorometric analysis. In a preliminary examination of this principle, we have demonstrated that we can correctly identify upregulation of three secreted proteins, following stimulation of a breast carcinoma cell line with various steroids. Our method should be suitable for high‐density microarray analysis of proteins, captured by specific monoclonal antibodies or other binding reagents. Copyright © 2000 John Wiley & Sons, Ltd.