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The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1.Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1.A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmuno-precipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb.An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1–1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1–1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1–1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1–1 effectively inhibited the binding of gp 120 to soluble CD4 in ELISA.These results suggest that the epitope recognized by S1–1 is con-formational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1–1 might have a role to play in vaccine development or passive immunotherapy.