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.
KeywordsHigh Performance Liquid Chromatography Platinum Bromide Electrophoresis Immobilization
G.F. Blackburn, H.P. Shah, J.H. Kenten, et al. Electrochemiluminescence detection for development of immunoassays and DNA-probe assays for clinical diagnostics. Clin. Chem.
37:1534 (1991).PubMedGoogle Scholar
E.O. Major, K. Amemiya, C.S. Tornatore, S.A. Houff, and J.R. Berger. Pathogenesis and molecular biology of progressive multifocal leukoencephalo-pathy, the JC virus-induced demyelinating disease of the human brain. Clin. Microbiol Rev.
5:49 (1992).PubMedGoogle Scholar
C.Tornatore, J.R. Berger, S.A. Houff, et al. Detection of JC virus DNA in peripheral lymphocytes from patients with and without progressive multifocal leukoencephalopathy. Ann. Neurol.
31:454 (1992).PubMedCrossRefGoogle Scholar
A.M. Wang, M.V. Doyle, and D.F. Mark. Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA
86:9717 (1989). [Erratum,. 87:2865 (1990)]PubMedCrossRefGoogle Scholar
B. van Gemen, T. Kievits, R. Schukkink, et al. Quantification of HIV-1 RNA in plasma using NASBATM during HIV-1 primary infection. J.Virol. Meth.
43:177 (1993).CrossRefGoogle Scholar
J. DiCesare, B. Grossman, E. Katz, et al. A high-sensitivity electrochemi-luminescence-based detection system for automated PCR product quantitation. BioTechniques
G. Gilliland, S. Perrin, K. Blanchard, and H.F. Bunn. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA
87:2725 (1990).PubMedCrossRefGoogle Scholar
P.D. Siebert, and J.W. Larrick. PCR Minics: competitive DNA fragments for use as internal standards in quantitative PCR. BioTechniques
14:244 (1993).PubMedGoogle Scholar
M. Piatak, Jr., K-C. Luk, B. Williams, and J.D. Lifson. Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. BioTechniques
14:70 (1993).PubMedGoogle Scholar
R. Frisque, G. Bream, and M. Canulla. Human polyomavirus JC virus genome. J. Virol.
51:458 (1984).PubMedGoogle Scholar
J.H. Kenten, J. Casadei, S. Link, et al. Rapid electrochemiluminescence assays for polymerase chain reaction products. Clin. Chem.
37:1626 (1991).PubMedGoogle Scholar
J.H. Kenten, S. Gudibande, J. Link, J. Willey, and E. Major. An improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays for HIV-1 polymerase chain reaction products. Clin. Chem.
38: 873(1992).PubMedGoogle Scholar
J.M. Wages, L. Dolenga, and A.K. Fowler. Electrochemiluminescence detection and quantitation of PCR-amplified DNA. Amplifications
(Perkin Elmer) 10:1 (1993).Google Scholar
R.W. Cone, A.C. Hobson, M.-L. W. Huang. Coamplified positive control detects inhibition of polymerase chain reactions. J. Clin. Microbiol.
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