PBMC based assays. All PBMC neutralization assays are described as a/b/c where 'a' is the incubation time in hours following mixing of mAb with virus, 'b' is the absorption time in hours during which the cells are exposed to the mAb/virus mixture. Cells are then washed and 'c' is the culture time in days (all at 37uC and 7% CO 2 ). In this study results were obtained in 24/1/14, 1/2/7 and 1/24/14 formats. These are named 'extended incubation', 'short incubation' and 'extended absorption' assays respectively. The extended incubation assay, which was originally used for patient selection has been described previously [6, 11] . Briefly, virus stock is diluted in a five fold series from 1/2 to 1/6250 in culture medium (RPMI-1640 medium supplemented with 15% FCS and 200 U/ml IL-2) to establish the titer (-log10 if the dilution at which 50% infection is achieved). A titer below 1 constitutes poor growth of the virus and the experiment is discarded. Ninety ml of each virus dilution are mixed with 5 ml of plasma or 50 mg mAb. In assays testing neutralization by plasma the mixture is complemented with 5 ml culture medium to give a final 1 in 20 dilution of plasma. When testing mAb, the mixture is complemented with 5 ml flow through (IgG was removed from HIV-1 negative plasma using a Protein G column [GE Healthcare Europe GmbH, Belgium]) to give 50 mg/ml of mAb. After the incubation phase 20 ml of each plasma or mAb/ virus mix are first dispensed in quadruplicate into flat bottom 96well microplates and 75,000 PBMC in 100 ml culture medium are added to each well. Plates are then left in a CO 2 incubator at 37uC during the absorption phase (b). Afterwards, cells are washed three times by centrifugation at 2000 rpm for 10 minutes, the supernatant is aspirated and 180 ml fresh culture medium are added to the cells. When cultured for 14 days, 125 ml of the medium is aspirated and replaced with 135 ml fresh culture medium. After c days, 200 ml of the supernatant are mixed with 50 ml Nonidet P40 (0.25% in PBS; Fluka, Sigma-Aldrich, Puurs, Belgium) to disrupt virions and this mixture is analyzed for the presence of HIV p24 antigen. As a control, pooled plasma of 100 HIV-1 negative donors are tested in parallel. For the short incubation and extended absorption phase assays, times of incubation are appropriately adjusted: 1/2/7 and 1/24/14 respectively. Neutralization activities are presented as the percentage reduction in infectious titer of a virus isolate following incubation with patient plasma or mAb relative to its titer following incubation with HIV-1 negative control plasma. Virus titers were calculated by the method of Reed and Muench [12] . An 80% reduction in titer was considered significant. By extending the usual one hour absorption phase of the extended and short incubation PBMC assay to 24 hours (1/24/14 format) we aimed to reproduce the conditions of the cell line based assays where the mAbs remain during the entire absorption and culture phase.