Streptavidin poly hrp 40

ISF Aβ concentrations were assessed using sandwich ELISAs as described (13 (link)). Briefly, a mouse-anti-Aβ₄₀ antibody (mHJ2) was used to capture and a biotinylated central domain antibody (mHJ5.1) was used to detect, followed by streptavidin-poly-HRP-40 (Fitzgerald Industries, Concord, MA). All ELISA assays were developed using Super Slow ELISA TMB (Sigma, St. Louis, MO) and absorbance read on a Bio-Tek Epoch plate reader (Winooski, Vermont) at 650nm. The standard curve for each assay utilized synthetic human Aβ_1-40 peptide (American Peptide, Sunnyvale, CA).

The concentration of tau and pTau was quantified as previously described ^{22 (link)} by sandwich ELISA using Tau-5 (in-house antibody) as the coating antibody and human-specific biotinylated HT7 for detection for tau ELISA and using HJ14.5 (in-house p.Thr181-tau antibody) as the coating antibody and human-specific biotinylated AT8 for detection for pTau ELISA. Briefly, 96-well halfarea plates were coated with 20 μg/mL of either HJ14.5 or Tau-5 antibody and incubated at 4°C overnight. The next day, the plate was blocked in 3% BSA (RPI Corp.) in PBS for 1 hour at 37°C. Next, peptide standards and samples were diluted in sample buffer (0.25% BSA/PBS, 1× protease inhibitor, 300 mM Tris pH 7.4, PBS), loaded onto the plate, and incubated at 4°C overnight. On the third day, 0.3 μg/mL of biotinylated AT8 for pTau ELISA (Thermo Fisher Scientific, MN1020B) or biotinylated HT7 for tau ELISA (Thermo Fisher Scientific, MN1000B) was applied to the plate for 1.5 hours at 37°C, and then Streptavidin-poly-HRP-40 (1:10,000 for tau and 1:6,000 for pTau) (Fitzgerald) was applied for 1.5 hours at room temperature. TMB Superslow Substrate solution (MilliporeSigma) was added and the plates were read at 650 nm on a BioTek plate reader after developing for 30 minutes at room temperature. All samples were run in duplicate.