Incubation at pH 2.5 induced artifactual ELISA signal has a lower affinity for Aβ than authentic anti-Aβ antibodies in competition assays. Sera from unvaccinated mice (panel A) or mice vaccinated with Aβ 1–42 peptide (panel B), were diluted 1:1000 and preincubated at either pH 7 or pH 2.5. After centrifugation, neutralization and reconstitution of sera volume, the sera were then incubated with human Aβ1–42 peptide, at increasing concentrations at 37°C for 60 minutes. Because the Aβ was initially dissolved in DMSO, some samples were also incubated with 2% DMSO as control. The ELISA assay was then performed as described in methods with no further separation of Aβ and antibody fractions. ** P < 0.001 compared to pH 2.5 without Aβ preincubation.