Amyloid beta (Aβ) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Aβ oligomers (amyloid β-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Aβ oligomers and fibrils but not the physiologically prevalent Aβ monomer. Discrimination derived from an epitope found in assemblies of Aβ1–28 and ADDLs but not in other sequences, including Aβ1–40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Aβ assemblies, these antibodies provide tools by which pathological Aβ assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.