2.2.1. Viral Load (VL), p24, and AlphaLISA Assays

All cultured supernatants were initially tested using commercially available FDA-approved nucleic acid and p24 antigen assays. All VL values were determined using an in-house Abbott RealTime HIV-1 assay on the m2000 system (Abbott Molecular, Des Plaines, IL, USA) for automated extraction of viral RNA from supernatant with a detection limit of 50 HIV-1 RNA copies/mL [32]. The HIV-1 p24 Antigen capture assay (PerkinElmer Life Sciences, Boston, MA, USA) was used to measure supernatant virus levels and to monitor HIV appearance in culture supernatants at various times post-infection for virus isolation. Analytical p24 value was determined via the least squares fit to the standard curve at an absorbance equal to the cutoff defined by the manufacturer (i.e., mean negative control O.D. + 0.050, LOD 3.5 pg/mL). The p24 antigen was also evaluated using the AlphaLISA assay [33]. In using each test kit, a serial 10-fold dilution was performed in base matrix HIV negative diluent (SeraCare Inc., Milford, MA, USA) in siliconized Eppendorf tubes prior to testing. All conventional assays were performed in accordance with the manufacturer’s instructions.