A1 Refereed original research article in a scientific journal
A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2
Authors: Talha SM, Salminen T, Swaminathan S, Soukka T, Pettersson K, Khanna N
Publisher: ELSEVIER SCIENCE BV
Publication year: 2011
Journal: Journal of Virological Methods
Journal name in source: JOURNAL OF VIROLOGICAL METHODS
Journal acronym: J VIROL METHODS
Number in series: 1
Volume: 173
Issue: 1
First page : 24
Last page: 30
Number of pages: 7
ISSN: 0166-0934
DOI: https://doi.org/10.1016/j.jviromet.2011.01.001
Abstract
This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1 env. incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu(3+) chelate or with nanoparticles doped with the Eu(3+) chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu(3+) chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios. (C) 2011 Elsevier B.V. All rights reserved.
This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1 env. incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu(3+) chelate or with nanoparticles doped with the Eu(3+) chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu(3+) chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios. (C) 2011 Elsevier B.V. All rights reserved.
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