Electrochemiluminescence-Based Detection System for the Quantitative Measurement of Antigens and Nucleic Acids: Application to HIV-1 and JC Viruses
A system for quantitative measurements based on electrochemiluminescence (ECL) has recently been developed. ECL is the generation of light through a series of chemical reactions at an electrode surface using a label that is a chelate of ruthenium(II) tris(2,2′-bipyridine) (Rubpy). ECL is ideally suited for analytic procedures involving antigens or nucleic acids because of the precision, sensitivity, and accuracy of the system. In addition, assay formats which eliminate wash steps and have rapid kinetics have been developed.
Applications of the ECL system which produce significant advantages over conventional methods are DNA probe assays and the quantitation of PCR products. Assay measurements produce a linear log-log plot (signal vs. concentration of analyte), facilitating quantitation over the wide dynamic range. Several assay formats have been developed. Sandwich hybridizations have been performed using biotinylated capture probes for the immobilization of analyte onto magnetic beads. Biotinylated primers can be used for this purpose, in polymerase chain reaction (PCR) amplification. The biotinylated PCR elongated strand or capture probe was bound to streptavidin-coated magnetic beads for detection by a Rubpy-labeled oligo probe. Oligonucleotide probe assays were performed in one step, 15-minute incubation time test formats. Experimental data indicated a sensitivity of 105 molecules per 10 μl sample of HIV-1 gag gene DNA and a five-order of magnitude linear dynamic range. The sensitivity and quantitative efficiency of the ECL system was demonstrated. The application of this system to the quantitation of the input copy number in competitive PCR amplification, using internal standards of JC virus, is also discussed.
KeywordsPolymerase Chain Reaction Reaction Probe Assay Quantitative Polymerase Chain Reaction Analysis Sandwich Hybridization Size Polymerase Chain Reaction Product
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