Amyloid-β (Aβ) deposits are important pathological hallmarks of Alzheimer's disease (AD). Aβ aggregates into fibrils; however, the intermediate oligomers are believed to be the most neurotoxic species and, therefore, are of great interest as potential biomarkers. Here, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for Aβ oligomers by using the same capture and (labeled) detection antibody. The ELISA predominantly recognizes relatively small oligomers (10-25 kDa) and not monomers. In brain tissue of APP/PS1 transgenic mice, we found that Aβ oligomer levels increase with age. However, for measurements in human samples, pretreatment to remove human anti-mouse antibodies (HAMAs) was required. In HAMA-depleted human hippocampal extracts, the Aβ oligomer concentration was significantly increased in AD compared with nondemented controls. Aβ oligomer levels could also be quantified in pretreated cerebrospinal fluid (CSF) samples; however, no difference was detected between AD and control groups. Our data suggest that levels of small oligomers might not be suitable as biomarkers for AD. In addition, we demonstrate the importance of avoiding HAMA interference in assays to quantify Aβ oligomers in human body fluids. © 2012 Elsevier Inc. All rights reserved.