Sa hrp

Anti-Aβ₄₂ antibody (Invitrogen 2μg/ml) was used as the coating antibody, and synthetic Aβ₄₂ was used as a standard. The medium samples, as well as the standards, were denatured in 0.5% sodium dodecyl sulfate (SDS) at 90 °C for 5min, followed by a 1:10 dilution (in order to lower the SDS concentration to 0.05% SDS). Biotinylated mAb4G8 (Nordic Biosite, 80070) was used as detection antibody, followed by incubation with SA-HRP (1:2000 Mabtech) for 1h at RT.

ELISpot plates (Millipore, Bedford, MA) were precoated with capture antibody (5 μg/ml) against IFN-γ (Mabtech, Cincinnati, OH) in 1× PBS and stored overnight at 4°C. Plates were washed five times in PBS and blocked with RPMI 1640 (Cellgro, Manassas, VA) containing 10% bovine serum albumin (Sigma-Aldrich) for 1 hour at 37°C in a humidified incubator followed by three washes in PBS. Responding PBMCs (3.0 × 10⁵ cells) were added to each well in 100 μl of complete RPMI 1640 supplemented with 10% pooled naïve monkey serum and l-glutamine, penicillin/streptomycin, and Hepes buffer (Invitrogen). The responding cells were cultured with an equal number of irradiated stimulating cells, medium alone, or PHA at 1 μg/ml (Sigma-Aldrich). After 48-hour incubation at 37°C, the plates were washed and biotinylated detection antibodies against IFN-γ (Mabtech, Cincinnati, OH) were added. The plates were then incubated at room temperature for 2 hours, washed with PBS, and incubated with SA-HRP (Mabtech) in PBS for 1 hour at room temperature, followed by an additional five washes. Trimethylboron (TMB; Moss, Pasadena, MD) was used to develop the spots. Development was done until distinct spots appeared. Plates were analyzed with an ELISpot image analyzer (Cellular Technology, Cleveland, OH).