Incubation at pH 2.5 induced increase in anti-Aβ ELISA signal is present not only in sera from vaccinated mice, but also untreated mice. Sera were collected from APP transgenic mice vaccinated (Vax +) with Aβ1–42 (panel A) and nonTg unvaccinated (Vax -) mice (panel B; normal serum), preincubated with dissociation buffer at either pH 7 (open bars) or pH 2.5 (solid bars) at room temperature for 20 minutes and centrifuged through 10,000 MW filters, brought to neutral pH and reconstituted to the original serum dilution volume. The sera were then incubated on ELISA plates coated with one of the following at 5 μg/ml; human Aβ1–42, phosphate buffered saline (PBS; a "no protein" control), bovine serum albumin (BSA), recombinant alpha-synuclein (a-SYN) or Aβ peptide amino acids 11–20. ELISA assays were completed as described in methods and the optical density (OD) at 450 nm used to estimate the amount of antibody binding to the ELISA plate. Note different Y-axis scales in panels A and B. Results are presented as mean ± SEM. ** P < 0.001 compared to pH 7.0.