Human βamyloid 1 40 elisa kit wako 2

Sandwich ELISA was used to quantify Aβ_1-40 and Aβ_1-42 levels using Human β Amyloid (1-40) ELISA Kit Wako II and a Human/Rat β Amyloid (1-42) ELISA Kit Wako, High-Sensitive according to the manufacturer’s instructions (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Microplates were pre-coated with monoclonal BAN-50 (IgG, anti-Aβ1-16) and sequentially incubated with 25μL of samples followed by horseradish-peroxidase-conjugated BA27 (anti-Aβ_1-40 (Fab’)2, specific for Aβ₄₀) or BC05 (anti-_Aβ35-43 (Fab’)2, specific for Aβ_42/43) [39 (link)]. Sensitivities were 0.019 pmol/L (assay range 1.0–100 pmol/L) in the Aβ_1-40 assay and 0.06 pmol/L (assay range 0.01–20.0 pmol/L) in the Aβ_1-42 assay. Intra- and inter-assay coefficients of variation were less than 10% in both assay systems.

To measure the amount of Aβ_1–40 or Aβ_1–42 in the brain tissue, a tris-buffered saline (TBS)-extracted fraction and a formic acid (FA)-extracted fraction were prepared according to the following procedures as described previously [32, 33 (link)]. To measure the amount of internalized Aβ_1–42 remaining in MG6 cells, MG6 cells were collected and lysed in RIPA sample buffer (Wako) with protease inhibitor cocktail (Wako). The amounts of Aβ_1–40 or Aβ_1–42 in FA-extracted fraction, TBS-extracted fraction and cell lysate were measured using Human βAmyloid (1–40) ELISA Kit Wako II (FUJIFILM Wako, Osaka, Japan) and human Aβ₄₂-specific ELISA kit (Thermo Fisher).