Simple diffusion-constrained immunoassay for p24 protein with the sensitivity of nucleic acid amplification for detecting acute HIV infection

Abstract

Nucleic acid amplification techniques have become the mainstay for ultimate sensitivity for detecting low levels of virus, including human immunodeficiency virus (HIV). As a sophisticated technology with relative expensive reagents and instrumentation, adoption of nucleic acid testing (NAT) can be cost inhibited in settings in which access to extreme sensitivity could be clinically advantageous for detection of acute infection. A simple low cost digital immunoassay was developed for the p24 capsid protein of HIV based on trapping enzyme-labeled immunocomplexes in high-density arrays of femtoliter microwells and constraining the diffusion of the enzyme-substrate reaction. The digital immunoassay was evaluated for analytical sensitivity for HIV capsid protein p24, and compared with commercially available NAT methods and immunoassays for p24, including 4th-generation antibody/antigen combo assays, for early detection of HIV in infected individuals. The digital immunoassay was found to exhibit 2000-3000-fold greater analytical sensitivity than conventional immunoassays reactive for p24, and comparable sensitivity to NAT methods. Assaying serial samples from 10 HIV-infected individuals, the digital immunoassay detected acute HIV infection as early as NAT methods, and 7-10 days earlier than conventional immunoassays. Comparison of assay results between the digital immunoassay and a quantitative NAT method from HIV infected serum exhibited a linear correlation R(2)>0.99. The data indicate that by constraining diffusion of the signal generation step of a simple sandwich immunoassay and enabling the digital counting of immunocomplexes, dramatic improvements in sensitivity to virus can be obtained to match the sensitivity of NAT at a fraction of the cost.

HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

“…To overcome this limitation, we optimized the digital immunoassay protocol, detailed in Methods, for the rapid and sensitive detection of HIV gag p24 protein in latently infected CD4 + T cell lysates prepared from ART-suppressed HIV + individuals and applied this approach to assess proviral reactivation and immune-mediated cell kill. Robust linearity in detecting p24 in CD4 + T cell lysates from aviremic HIV + individuals was demonstrated with an analytical limit of detection (LOD) of 5 fg/ml ( Figure 1A), corresponding to 125,000 p24 copies/ml or 18,000 copies/144 μl assay input and, thus, about 9 viruses/144 μl input (29,30). The optimized methods extended the sensitivity LODs for HIV p24 in cell lysates ( Figure 1B).…”

“…New ELISA technologies support increased detection of HIV gag p24 in plasma, serum, and cultured cell medium; however, these methods show decreased sensitivity in cell lysates due to matrix interference and in plasma and serum of chronically infected HIV + subjects due to immune complex formation after seroconversion (29,30). Here, we report optimized methods that enable sensitive and rapid detection of subfemtomolar cell-associated HIV gag p24 protein in HIV-infected CD4 + T cells from ART-suppressed individuals.…”

“…The fully automated Quanterix HD-1 analyzer for single molecule detection has been reported previously (29)(30)(31). In this study, cell lysates were prepared by adding a final concentration of 1% Triton X-100/PBS to 2 × 10 6 cells.…”

“…Recent studies have reported the use of a digital immunoassay to measure HIV gag p24 protein from serum or plasma of acutely infected HIV + individuals or cultured media of HIV-infected CD4 + T cells (29)(30)(31). Despite these advances, commercial methods encounter matrix issues and lack the same degree of sensitivity in quantifying cell-associated viral p24 protein from aviremic HIV + individuals.…”

Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection

“…Previously, we reported a prototype digital immunoassay for p24 that used an early version of singlemolecule-array (Simoa) technology (11,12 ). The assay was a bead-based ELISA requiring 7-8 h to complete a 96-well plate.…”