Alliance hiv p24 antigen elisa kit

Beta-lactamase-Vpr (BlaM-Vpr)-containing HIV-1 particles were generated to assay HIV-1 fusion, as described by Cavrois et al.^{28 (link)}. The two HIV-1 proviral clones used, which expressed either the X4 Env from HIV-1 NL4-3 or the R5 Env from HIV-1 JR-FL, were a kind gift from Dr. Bernard Lagane (CPTP, Toulouse, France), and have been reported previously^{57 (link)}. These proviral clones, which are derived from pNL4-3, carry a luciferase reporter gene in place of the nef gene, and differ in a fragment encompassing the env gene between positions 6113 and 8797. To produce BlaM-Vpr HIV-1 particles, 8 × 10⁶ HEK 293 T cells in 162 cm² culture flasks were cotransfected using the calcium phosphate-DNA co-precipitation method with 60 μg proviral DNA, 20 μg pCMV-BlaM-Vpr plasmid, and 10 μg pAdVAntage vector (Promega), which increases transient protein expression. The culture media was replaced 18 h post-transfection, and supernatants containing viral particles were harvested at 48 h. The supernatants were clarified at low speed (1460 g) and then ultracentrifuged at 23,000 g for 90 min at 4 °C. The pelleted viruses were resuspended in DMEM 10% FBS at a final 50x concentration, quantified for their content in HIV-1 Gag p24 antigen (Alliance HIV P24 antigen ELISA Kit from PerkinElmer), and stored at −80 °C before use.

BlaM-vpr-containing viral clones were prepared in HEK 293T cells as described [110 (link)]. Briefly, 1.5 x 10⁷ cells in 162 cm² culture flasks were cotransfected using the calcium phosphate-DNA coprecipitation method with 60 μg proviral DNA, 20 μg pCMV-BlaM-vpr plasmid and 10 μg pAdvantage vector (Promega). Forty-eight hours post-transfection, culture supernatants containing the viral particles were clarified at low speed and then ultracentrifuged at 72,000 g for 90 min at 4°C. The pelleted viruses were then resuspended in DMEM, measured for their content in HIV-1 Gag p24 antigen (Alliance HIV P24 antigen ELISA Kit from PerkinElmer) and stored at -80 °C before use.
Fifty ng Gag p24 of BlaM-vpr-containing viruses were exposed to 2 x 10⁵ T-cells or 1.5 x 10⁵ MDMs, which were detached from culture flasks with Cellstripper (Corning), as previously described [42 (link)]. Then, cells were incubated for 2 h with the CCF2/AM dye (using the CCF2-AM loading kit from Invitrogen). Loading of the A3.01 T cells with CCF2 was performed in the presence of the AlexaFluor 647-conjugated anti-Flag mAb M2 at a 1:100 final dilution. Enzymatic cleavage of CCF2 by β-lactamase in the target cells was analyzed by flow cytometry (FACSCanto, BD Biosciences).