For TfR ELISA, 96-well half-area plates (Corning Inc., Corning, NY) were coated overnight at 4 °C with 500 ng/mL of mouse TfR protein (recombinantly prepared in the lab). This was followed by 2-h incubation with ELISA blocking buffer (1% BSA in PBS) at RT.
For Aβ ELISA, 96-well half-area plates (Corning Inc., Corning, NY) were coated overnight at 4 °C with 1 μg/mL of rabbit polyclonal antibodies detecting the C-terminal of Aβ (44–136, Thermo Scientific, Waltham, MA) (our in-house experiments show that it detects both Aβ40 and 42) or Aβ1-42 (700,254, Thermo Scientific, Waltham, MA). This was followed by 2-h incubation with ELISA blocking buffer (1% BSA in PBS) at RT, followed by additional 2-h incubation with 1 nM of Aβ1-40 monomers (Innovagen, Lund, Sweden) or 1 nM of Aβ1-42 aggregates, prepared as previously described [50 (link)]. For both ELISAs, serial dilutions of the recombinant proteins were prepared in ELISA incubation buffer (0.1% BSA, 0.05% Tween-20 in PBS) and added to the plates. The plates were incubated for 2 h at RT, followed by the addition of a goat anti-mouse HRP secondary antibody (Sigma Aldrich, Stockholm, Sweden) for 1 h at RT. K-blue aqueous TMB (Neogen Corp, Lexington, KY) was used for signal development and 1 M H2SO4 as the stop solution. Using a Spark multimode microplate reader (Tecan, Männedorf, Switzerland), absorbance was measured at 450 nm.
For Aβ ELISA, 96-well half-area plates (Corning Inc., Corning, NY) were coated overnight at 4 °C with 1 μg/mL of rabbit polyclonal antibodies detecting the C-terminal of Aβ (44–136, Thermo Scientific, Waltham, MA) (our in-house experiments show that it detects both Aβ40 and 42) or Aβ1-42 (700,254, Thermo Scientific, Waltham, MA). This was followed by 2-h incubation with ELISA blocking buffer (1% BSA in PBS) at RT, followed by additional 2-h incubation with 1 nM of Aβ1-40 monomers (Innovagen, Lund, Sweden) or 1 nM of Aβ1-42 aggregates, prepared as previously described [50 (link)]. For both ELISAs, serial dilutions of the recombinant proteins were prepared in ELISA incubation buffer (0.1% BSA, 0.05% Tween-20 in PBS) and added to the plates. The plates were incubated for 2 h at RT, followed by the addition of a goat anti-mouse HRP secondary antibody (Sigma Aldrich, Stockholm, Sweden) for 1 h at RT. K-blue aqueous TMB (Neogen Corp, Lexington, KY) was used for signal development and 1 M H2SO4 as the stop solution. Using a Spark multimode microplate reader (Tecan, Männedorf, Switzerland), absorbance was measured at 450 nm.