Innotest amyloid 1 42 elisa kit

The amount of t-tau was quantitatively analyzed using the commercially available enzyme-linked immunosorbent assay (ELISA) INNOTESThTAU-Ag kit from Fujirebio. The amount of p-tau phosphorylated at Thr181 was measured with the INNOTEST PHOSPHO-TAU(181P) ELISA kit from Fujirebio. The amount of Aβ was quantified using the INNOTEST ß-AMYLOID (1–42) ELISA kit from Fujirebio. AD cut-offs for t-tau and Aβ were 450 pg/mL according to data from the rpAD study.

A 96-well plate was coated overnight with goat anti-apoE (K74190G, Meridian Life Sciences, Memphis, TN, diluted 1:3000 in PBS) at 4 °C, followed by washing with PBS containing 0.05% Tween20 (PBST) and 2-h blocking with Odyssey blocking buffer (LI-COR Biosciences, Bad Homburg vor der Höhe, Germany) diluted 1:1 in PBS. After washing, wells were incubated with the Aβ-apoE protein samples (added in duplicate) diluted 200 times in sample diluent (INNOTEST ß-Amyloid (1-42) ELISA kit; Fujirebio, Ghent, Belgium) for 2 h at RT, while shaking at 600 RPM. Wells were then washed and incubated for 1 h at RT with biotinylated anti-Aβ antibody (mouse-α-Aβ clone 4G8, Biolegend, San Diego, CA; cat. 800701, diluted 1:2500 in PBS containing 1% BSA), while shaking at 600 RPM. Subsequent washing was followed by 30-min incubation with streptavidin-HRP (ThermoFisher, Waltham, MA, diluted 1:60000 in PBST), at RT, with shaking at 600 RPM. After the final washing step, TMB solution (Sigma-Aldrich) was added as a substrate. The reaction was stopped with 1 M H₂SO₄. Optical density (OD) values were measured at 450 nm on a Tecan Infinity F50 plate reader.