An ultrasensitive planar array p24 Gag ELISA to detect HIV-1 in diverse biological matrixes

Abstract

Human immunodeficiency virus-1 (HIV-1) persistence in the presence of antiretroviral therapy (ART) has halted the development of curative strategies. Measuring HIV persistence is complex due to the low frequency of cells containing virus in vivo. Most of the commercially available assays to date measure nucleic acid. These assays have the advantage of being highly sensitive and allow for the analysis of sequence diversity, intactness of the HIV genome or evaluation of diverse RNA species. However, these assays are limited in evaluating translational competent viral reservoirs. In here, we developed an ultrasensitive p24 ELISA that uses the Simoa planar array technology that can detect HIV-1 virions and HIV-1 infected cell with limit of detection similar to nucleic acid assays. Furthermore, the assay is optimized to measure very low levels of p24 in different biological fluids without a major loss of sensitivity or reproducibility. Our results demonstrate that the ‘homebrew’ planar p24 ELISA immunoassay is a broadly applicable new tool to evaluate HIV persistence in diverse biological fluids and cells.

The latency-reversing agent HODHBt synergizes with IL-15 to enhance cytotoxic function of HIV-specific T cells

“…Ultrasensitive p24 ELISA. Supernatants were collected for p24 detection using an ultrasensitive ELISA (SP-X technology) as described by Levinger et al (71). As treatment conditions of the HIVE assays were plated across multiple wells of 96-well plates, multiple independent supernatant measures were made for each.…”

IL-27 induces IFN/STAT1-dependent genes and enhances function of TIGIT+ HIVGag-specific T cells

“…In these culture conditions we did not detect HIVp24 production. HIVp24 was detected after 4 days culture of PBMCs stimulated with CD3 and CD28 mAbs used as positive control ( Figure S5 , Levinger et al., 2021 ).…”

“…As control, PBMCs were stimulated with CD3/CD28 mAbs (T cell TransAct catalog # 130-111-160, Miltenyi Biotec, Germany) overnight and during three and four days. Supernatants were colleted for p24 detection using an ultra-sensitive ELISA (SP-X technology) as described by ( Levinger et al, 2021 ). Briefly, plates coated with 1 μg/mL capture antibody (HIV-018-48,303, Capricorn Products LLC, ME) were incubated with 50 μL of each culture condition in triplicates for 2 hours on a shaker.…”

QuickFit: A high-throughput RT-qPCR-based assay to quantify viral growth and fitnessin vitro

“…Longitudinal samples are subjected to either ELISA-or RT-qPCR-based methods to measure viral antigens or genetic material, respectively (22). However, these approaches are time-consuming, expensive, and have varying degrees of sensitivity (23,24), which leads to inconsistent reports of growth rates in the literature (25)(26)(27).…”