Mouse anti aβ clone 4g8

Aβ(M1–37- GGGK(Btn)) standards were prepared in diluent (TBS+0.1% Tween-20+1% BSA) in a range of concentrations from 20 nM to 312 pM. Samples were diluted as necessary in this same diluent. Pre-blocked Streptavidin Coated High Capacity plates (clear, 96-well, Pierce) were washed 2x with TBS-T (TBS + 0.1% Tween-20) before addition of standards and any samples. Biotinylated material was captured by streptavidin at room temperature for 2 hours. Plates were washed three times with TBS-T. 100 μL of mouse anti-Aβ clone 4G8 (1:2000 in diluent, Biolegend, 800702) was added to each well and incubated at room temperature for one hour. Following 3x TBS-T washes, each well was treated with 100 μL of goat anti-mouse IgG HRP conjugate (1:4000 in diluent, ThermoFisher, A-10668) for 30 minutes at room temperature. Plates were washed four times with TBS-T before addition of 50 μL TMB (ThermoFisher, 34028). Wells were allowed to develop until saturation and then quenched with 50 μL of 2M H₂SO₄. The absorbance of each well at 450 nm was then measured using a Tecan Plate Reader. The standard curve was fitted to 4-parameter logistics curve by Solver in Excel and used to calculate concentration of biotinylated Aβ in present samples.