title: Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.
abstract: Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Mohan Kumar
authors.initials: MK
authors.lastname: Haleyur Giri Setty
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Jikun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Prerna
authors.initials: P
authors.lastname: Mahtani
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Panhe
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Bingchen
authors.initials: B
authors.lastname: Du
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Viswanath
authors.initials: V
authors.lastname: Ragupathy
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Krishnakumar
authors.initials: K
authors.lastname: Devadas
authors.affiliation: Laboratory of Molecular Virology, CBER , FDA, Silver Spring, Maryland.
authors.forename: Indira K
authors.initials: IK
authors.lastname: Hewlett
mesh_terms: D005063:Europium / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D053758:Nanoparticles / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D005063:Europium