Galacton star substrate

We previously adapted a fusion assay based on α complementation of β-galactosidase (β-Gal) (11 (link)). In this assay, receptor-bearing cells expressing the omega peptide of β-Gal are mixed with cells coexpressing envelope glycoproteins and the α peptide of β-Gal, and cell fusion leads to complementation. Fusion is stopped by lysing the cells. Substrate was added (Galacton-Star substrate; Applied Biosystems, T1012), and luminescence was read after 1 hour at 500 ms on a Tecan M1000 Pro. Percent inhibition was quantified by comparing relative luminescence units (RLUs) at different concentrations of antibody to RLUs in the absence of antibody 100 × [1 − (luminescence at X background)/(luminescence in the absence of inhibitor − background)].

We produced viruses by transfection of 293T cells using GeneJuice (Novagen). Laboratory-adapted provirus R9 (NL4.3 derivative) was kindly provided by D. Trono. We obtained CD4⁺ TZM-bl cells through the AIDS Research and Reference Program and 293T cells from ATCC. All viruses were harvested 48 h post-transfection, filtered (0.2-mM filter, Pall Acrodisc), and normalized by p24 ELISA (Alliance HIV-1 p24 Antigen ELISA Kit; Perkin Elmer)
TZM-bl cells were seeded in 48-well plates at 4.5 × 10⁴ cells per well, 24 h before infection in 200 μl of high glucose DMEM (Gibco) 10% FBS. Neutralizing antibodies were incubated with 10 ng HIV-1 (p24 equivalents) in cell media for 30 min at 37°C before being added to cells. We washed the cells with PBS and lysed them 72 h postinfection with Galacton-Star lysis buffer. We transferred 20 μl of cell lysate to a 96-well plate for detecting β-galactosidase activity. One hundred microliters of reaction buffer [Galacton-Star substrate (Applied Biosystems, Bedford, MA) diluted 1:50] was added to 20 μl of lysate, and the light emission was measured in relative fluorescence units over 1 s in a microplate luminometer after 30 min incubation.