title: A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2.
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-1env, 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.
authors.affiliation: International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Sheikh M
authors.initials: SM
authors.lastname: Talha
authors.forename: Teppo
authors.initials: T
authors.lastname: Salminen
authors.forename: Sathyamangalam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry / Q000379:methods*; D015483:HIV Antibodies / Q000097:blood*; D015658:HIV Infections / Q000175:diagnosis* / Q000821:virology; D015497:HIV-1 / Q000276:immunology*; D015498:HIV-2 / Q000276:immunology*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics
chemical_list: D015483:HIV Antibodies; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus