# Development of an Ultrasensitive ELISA Assay for Evaluating HIV-1 Envelope Glycoprotein as a Marker for Targeted Activator of Cell Kill

## TL;DR

A new ultrasensitive ELISA test was developed to measure HIV-1 gp120, a key protein on infected cells, to help evaluate treatments targeting HIV.

## Contribution

Development of a highly sensitive and cost-effective ELISA assay for HIV-1 gp120 with validation in cell lysates and correlation with other HIV markers.

## Key findings

- The ELISA achieved a lower limit of quantitation (LLOQ) of 0.16 pM, comparable to the Simoa platform.
- The assay's antibody specificity was confirmed with LC-MS and validated using HIV-infected cell lysates.
- gp120 levels correlated with immunocytochemistry results, p24 levels, and viral RNA measurements.

## Abstract

The HIV-1 envelope glycoprotein gp120 is prominently exposed on the surface of both HIV-1 virions and infected host cells, serving as a key marker of infection. gp120 plays a pivotal role in viral entry by interacting with the primary receptor, CD4, on host cells. Therapeutic strategies targeting the HIV-1 reservoir, such as anti-gp120 antibodies that trigger antibody-dependent cellular cytotoxicity (ADCC) and chimeric antigen receptor T (CAR-T) cells, rely on the presence of gp120 on the surface of infected cells to exert their effects. Consequently, accurate monitoring of gp120 expression on infected cells is essential for evaluating the pharmacological efficacy of these interventions. In this study, a sensitive, specific, and inexpensive enzyme-linked immunosorbent assay (ELISA) for quantifying HIV-1 gp120 glycoprotein was developed using a selected pair of anti-gp120 antibodies. The assay achieved a lower limit of quantitation (LLOQ) of 0.16 pM, demonstrating sensitivity comparable to that of the digital single molecule array (Simoa) platform, which exhibited a LLOQ of 0.23 pM and requires specialized instrumentation. The binding specificity of the antibodies used in the novel assay was confirmed using liquid chromatography–mass spectrometry (LC-MS), and the assay was pharmacologically validated with lysates obtained from 2D10 and MOLT IIIB cell lines. Furthermore, treatment of HIV-infected human primary CD4+ T cells with a targeted activator of cell kill (TACK) compound significantly reduced gp120 concentration in CD4+ T cell lysate compared to controls. The gp120 marker from infected cell lysates correlated with the number of gp120-positive cells detected by immunocytochemistry, as well as with HIV-1 p24 levels and cell-associated viral RNA measurements. In summary, a novel, simple, and sensitive HIV-1 gp120 ELISA has been developed and validated. This assay holds potential for investigating HIV-1 persistence and evaluating the efficacy of therapeutic agents targeting infected cells.