Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes

Mélanie Merbah; Sayali Onkar; Jean-Charles Grivel; Christophe Vanpouille; Angélique Biancotto; Lydia Bonar; Eric Sanders-Buell; Gustavo Kijak; Nelson Michael; Merlin Robb; Jerome H Kim; Sodsai Tovanabutra; Agnès-Laurence Chenine

doi:10.1016/j.jviromet.2016.01.009

Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-1 subtypes

J Virol Methods. 2016 Apr:230:45-52. doi: 10.1016/j.jviromet.2016.01.009. Epub 2016 Jan 22.

Authors

Affiliations

¹ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: mmerbah@hivresearch.org.
² U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: sonkar@hivresearch.org.
³ Eunice Kennedy Shriver National Institute of Child Health and Human Development, Program of Physical Biology, National Institutes of Health, MD, USA. Electronic address: jgrivel@sidra.org.
⁴ Eunice Kennedy Shriver National Institute of Child Health and Human Development, Program of Physical Biology, National Institutes of Health, MD, USA. Electronic address: vanpouic@mail.nih.gov.
⁵ Center for Human Immunology, Autoimmunity, and Inflammation, National Institutes of Health, Bethesda, USA. Electronic address: biancoa@nhlbi.nih.gov.
⁶ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: lbonar@hivresearch.org.
⁷ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: esanders-buell@hivresearch.org.
⁸ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: gkijak@hivresearch.org.
⁹ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA. Electronic address: nmichael@hivresearch.org.
¹⁰ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: mrobb@hivresearch.org.
¹¹ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA. Electronic address: jerome.kim@ivi.int.
¹² U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: stovanabutra@hivresearch.org.
¹³ U.S. Military HIV Research Program, Walter Reed Army Institute of Research, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, MD, USA. Electronic address: achenine@hivresearch.org.

Abstract

The prevailing method to assess HIV-1 replication and infectivity is to measure the production of p24 Gag protein by enzyme-linked immunosorbent assay (ELISA). Since fluorescent bead-based technologies offer a broader dynamic range and higher sensitivity, this study describes a p24 capture Luminex assay capable of detecting HIV-1 subtypes A-D, circulating recombinant forms (CRF) CRF01_AE and CRF02_AG, which together are responsible for over 90% of HIV-1 infections worldwide. The success of the assay lies in the identification and selection of a cross-reactive capture antibody (clone 183-H12-5C). Fifty-six isolates that belonged to six HIV-1 subtypes and CRFs were successfully detected with p-values below 0.021; limits of detection ranging from 3.7 to 3 × 104 pg/ml. The intra- and inter-assay variation gave coefficient of variations below 6 and 14%, respectively. The 183-bead Luminex assay also displayed higher sensitivity of 91% and 98% compared to commercial p24 ELISA and a previously described Luminex assay. The p24 concentrations measured by the 183-bead Luminex assay showed a significant correlation (R=0.92, p<0.0001) with the data obtained from quantitative real time PCR. This newly developed p24 assay leverages the advantages of the Luminex platform, which include smaller sample volume and simultaneous detection of up to 500 analytes in a single sample, and delivers a valuable tool for the field.

Keywords: Detection; HIV-1; Multi-subtype; Multiplex assay; p24.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Biological Assay / methods
Enzyme-Linked Immunosorbent Assay* / standards
Fluorescent Antibody Technique, Indirect* / standards
HIV Core Protein p24 / immunology*
HIV Infections / virology
HIV-1* / classification
HIV-1* / immunology
Multiplex Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Sensitivity and Specificity

Substances

HIV Core Protein p24

Grants and funding

Z99 HD999999/Intramural NIH HHS/United States