Development and validation of a quantitation assay for fluorescently tagged HIV-1 virus-like particles

Sonia Gutiérrez-Granados, Laura Cervera, Francesc Gòdia, Jorge Carrillo, María Mercedes Segura

Research output: Contribution to journalArticleResearchpeer-review

30 Citations (Scopus)

Abstract

Upon expression, the Gag polyprotein of HIV-1 assembles spontaneously in the vicinity of the plasma membrane giving rise to enveloped virus-like particles (VLPs). These particulate immunogens offer great promise as HIV-1 vaccines. Robust VLP production and purification processes are required to generate VLPs of sufficient quality and quantity for both pre-clinical and clinical evaluation. The availability of simple, fast and reliable quantitation tools is critical to develop, optimize and monitor such processes. Traditionally, enzyme-linked immunosorbent assays (ELISA) are used to quantify p24 antigen concentrations, which reflect tightly virus particle concentrations. However, this quantitation technique is not only time-consuming, laborious and costly but it is also prone to methodological variability. As an alternative, the development and validation of a fluorescence-based quantitation assay for Gag VLPs is presented here. A Gag polyprotein fused to the enhanced green fluorescent protein was used for generation of fluorescent VLPs. A purified standard reference Gag-GFP VLP material was prepared and characterized in house. The method was validated according to ICH guidelines. The validation characteristics evaluated included accuracy, precision, specificity, linearity, range and limit of detection. The method showed to be specific for Gag-GFP. The fluorescence signal correlated well with p24 concentrations measured using a reference p24 ELISA assay. The method showed little variability compared to ELISA and was linear over a 3-log range. The limit of detection was ~10. ng of p24/mL. Finally, fluorescence-based titers were in good agreement with those obtained using transmission electron microscopy and nanoparticle tracking analysis. This simple, rapid and cost-effective quantitation assay should facilitate development and optimization of bioprocessing strategies for Gag-based VLPs. © 2013 Elsevier B.V.
Original languageEnglish
Pages (from-to)85-95
JournalJournal of Virological Methods
Volume193
Issue number1
DOIs
Publication statusPublished - 1 Oct 2013

Keywords

  • Fluorescently tagged virus
  • Fluorometry
  • Gag-GFP
  • HIV-1 VLPs
  • Quantitation assays
  • Virus-like particles (VLPs)

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