Despite some tests for detecting reservoirs of human immunodeficiency virus type 1 (HIV-1) capable of causing reinfection, there is a lack of methods to assess the extent of translationally competent and consequently biologically active HIV-1 reservoirs. In this work, we optimized an enzyme-linked immunosorbent assay (ELISA) for detecting the viral protein Nef. Nef is released from HIV-1 reservoirs, is present in various biological fluids, and contributes to HIV-1 pathogenicity. The optimized ELISA utilized biotinylated anti-Nef nanobody sdAb19, immobilized on the neutravidin-coated surface. Recombinant Nef (rNef) was detected in a buffer with a limit of the blank at 4.25 ng/ml and a limit of detection at 6.79 ng/ml. These values align with physiological Nef concentrations in the plasma of infected individuals. The quantitative range of the sdAb19-based ELISA spans from 10 ng/ml to 400 ng/ml. Notably, we observed a high limit of the blank when detecting rNef diluted in human serum. Through further optimization of the assay in complex biological fluids, our method could prove efficient in evaluating the size of pathologically relevant active HIV-1 reservoirs.
|
