Antibody Dependent Cellular Cytotoxic activity mediated by the patient plasma/sera and the HIVIG polyclonal IgG preparation was detected according to our modification of the previously described GranToxiLux (GTL) cell-mediated cytotoxicity procedure (23 (link),24 (link)). The assay was performed in 96-well plates as follows and outlined in Figure 1A. Infected and uninfected target cells were counted, washed, resuspended in R10 at 1×106 cell/ml, and labeled with a fluorescent target-cell marker (TFL4; OncoImmunin, Inc., Gaithersburg, MD) and a viability marker (NFL1; OncoImmunin, Inc.) for 15 minutes in a 37°C water bath as specified by manufacturer. After two washes using 10 ml of R10, viable cells were counted using a Guava PCA (Millipore, Billerica, MA) and adjusted to reach a final viable effector to viable target ratio of 30:1 or 10:1 when PBMC and NK cells were used as effector cells, respectively. Twenty-five μl of each effector and target cell suspension and 75 μl of GzB substrate (OncoImmunin, Inc.) were dispensed into each well of a 96-well V-bottom plate. After incubation for 5 minutes at room temperature (RT), 25μl of the appropriate antibody or IgG dilutions were added to the target/effector cell suspension and incubated for 15 minutes at RT. The plates were subsequently centrifuged for 1 minute at 300 × g, and incubated for 1 hour at 37°C and 5% CO2. After two washes with WB, cells were resuspended in 225μl of WB, placed at 4°C, and acquired directly from the assay plate with the LSRII (BD Bioscience, San Jose, California) within 6 hours using the High-Throughput-System (HTS, BD Bioscience, San Jose, California). A minimum of 2.5×103 and 5×103 events representing viable gp120-coated and infected target cells, respectively, was acquired for each condition. The signal for each fluorophore was detected using: 1) 640nm/40mW laser and 660/20 filter for TFL4; 405nm/50mW laser and 450/50 filter for NFL1; 3) 488nm/20mW laser and the combination of 505LP with 525/50 filters for the GzB substrate. Because of the spectral properties of the fluorescent molecules utilized in this panel, manual compensation of the signals was not required to analyze the data, as previously reported (24 (link)). Data analysis was performed using FlowJo 8.8.4 software. The investigators developed a dedicated analysis template that reflects the gating strategy illustrated in Figure 1B.