A single-cycle infectivity assay was used to measure the neutralization of luciferase-encoding virions pseudotyped with the desired HIV-1 Env proteins, as previously described [51] . Briefly, appropriate dilutions of the virion-containing culture supernatants were pre-incubated at 37uC for 1 h with mAbs at various concentrations. The virus-mAb mixtures were added to HOS-CD4-CCR5 cells and incubated for 3 days at 37uC. A similar protocol was used for supernatants screening using TZM-bl cells [45] . The cells were then lysed with Britelite reagent (Perkin-Elmer) and the relative light units in the cell lysates were determined on a luminometer microplate reader (Veritas, Turner Biosystems). The 50% inhibitory dose (IC50) was defined as the sample concentration at which relative luminescence units were reduced 50% compared to virus control wells. Some of the neutralization data for the reference mAbs b12, 2G12, 2F5 and 4E10 are taken from previous analyses [41, 42] carried out under identical standardized conditions to those used for the new mAbs reported here. A multi-cycle virus replication assay was used to measure the neutralization of replication competent luciferaseencoding viruses in 5.25.EGFP.Luc.M7 cells [52] . This cell line is a genetically engineered clone of CEMx174 that expresses multiple SIV and HIV-1 entry receptors (CD4, CCR5, CXCR4, GPR15/Bob) [53] . For the neutralization assay, 5000 tissue culture infectious dose 50 (TCID 50 ) of virus was incubated with multiple dilutions of test sample in triplicate for 1 hr at 37uC in a total volume of 150 ml in 96-well flat-bottom culture plates. A 100 ml suspension of cells (5610 5 cells/ml of growth medium containing 25 mg DEAE dextran/ml) was added to each well. Plates were incubated until approximately 10% of cells in virus control wells were positive for GFP expression by fluorescence microscopy (approximately 3 days). At this time, 100 ml of cell suspension was transferred to a 96-well white solid plate (Costar) for measurements of luminescence using the Britelite Luminescence Reporter Gene Assay System (PerkinElmer Life Sciences).