title: Proof of principle: an HIV p24 microsphere immunoassay with potential application to HIV clinical diagnosis.
abstract: The measurement of CD4 counts and viral loads on a single instrument such as an affordable flow cytometer could considerably reduce the cost related to the follow-up of antiretroviral therapy in resource-poor settings. The aim of this study was to assess whether the HIV-1 p24 antigen could be measured using a microsphere-based flow cytometric (FC) assay and the experimental conditions necessary for processing plasma samples. A commercial anti-p24 antibody pair from Biomaric was used to develop a p24 microsphere immunoassay (MIA) using HIV culture supernatant as the source of antigen. The ultrasensitive Perkin Elmer enzyme immunoassay (EIA) served as a reference assay. Quantification of HIV p24 using the heat-mediated immune complex disruption format described for plasma samples was feasible using the Biomaric MIA and applicable to a broad range of HIV-1 Group M subtypes. The inclusion of a tyramide amplification step was successful and increased the fluorescence signal up to 3 logs as compared with the MIA without amplification. The analytical sensitivity of this ultrasensitive Biomaric assay reached 1 pg/mL, whereas the ultrasensitive Perkin Elmer EIA was sensitive to less than 0.17 pg/mL. Our data indicate, for the first time, that the principle of p24 detection using the heat-denatured ultrasensitive format can be applied to FC.
authors.affiliation: Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp, Belgium. pondoa@itg.be
authors.forename: Pascale
authors.initials: P
authors.lastname: Ondoa
authors.forename: Chris
authors.initials: C
authors.lastname: Vereecken
authors.forename: Eugene Lekeawung
authors.initials: EL
authors.lastname: Asahchop
authors.forename: Amber
authors.initials: A
authors.lastname: Litzroth
authors.forename: Aziz
authors.initials: A
authors.lastname: Diallo
authors.forename: Katrien
authors.initials: K
authors.lastname: Fransen
authors.forename: Tandakha
authors.initials: T
authors.lastname: Dieye
authors.forename: Robin
authors.initials: R
authors.lastname: Ryder
authors.forename: Souleymane
authors.initials: S
authors.lastname: Mboup
authors.forename: Luc
authors.initials: L
authors.lastname: Kestens
mesh_terms: D005434:Flow Cytometry; D016655:HIV Core Protein p24 / Q000032:analysis*; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000276:immunology* / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008863:Microspheres; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D016655:HIV Core Protein p24