Mab20.1 antibody

For the determination of the Aβ levels, T-Per soluble fractions were loaded directly onto ELISA plates, whereas the formic acid supernatants (insoluble fractions) were diluted 1:20 in a neutralization buffer (1 M Tris base, 0.5 M NaH₂PO₄) before loading. MaxiSorp immunoplates (Nunc, Rochester, NY, USA) were coated with mAb20.1 antibody (Dr. William E. Van Nostrand, Stony Brook University, Stony Brook, NY, USA) at a concentration of 25 µg/ml in coating buffer (0.1 M Na₂CO₃, pH 9.6) and blocked with 3% bovine serum albumin. Standard solutions for both Aβ₄₀ and Aβ₄₂ were made in the antigen capture buffer (20 mM NaH₂PO₄, 2 mM EDTA, 0.4 M NaCl, 0.05% 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate, and 1% bovine serum albumin, pH 7.0) and loaded onto ELISA plates in duplicate. Samples were then loaded (also in duplicate) and incubated overnight at 4°C. Plates were then washed and probed with either horseradish peroxidase-conjugated anti-Aβ₄₀ (C49) or anti-Aβ₄₂ (D32) (Dr. Vitaly Vasilevko and Dr. David H. Cribbs, University of California Irvine, CA, USA) overnight at 4°C. The chromogen was 3,3',5,5'-tetramethylbenzidine, and the reaction was stopped by 30% phosphoric acid. The plates were read at 450 nm using a plate reader (Molecular Dynamics, Sunnyvale, CA, USA). The readings were then normalized to protein concentrations of the samples.

For determination of the Aβ levels, MaxiSorp immunoplates (Nunc, Rochester, NY) were coated with mAb20.1 antibody (Dr. William E. Van Nostrand, Stony Brook University, Stony Brook, NY) at a concentration of 25 μg/mL in coating buffer (0.1 mol/L Na₂CO₃, pH 9.6) and blocked with 3% bovine serum albumin. Standard solutions for both Aβ₄₀ and Aβ₄₂ were made in the antigen capture buffer (20 mmol/L NaH₂PO₄, 2 mmol/L EDTA, 0.4 mol/L NaCl, 0.05% 3-[(3-cholamidopropyl) dimethylammonio]propanesulfonate, and 1% bovine serum albumin, pH 7.0) and loaded onto ELISA plates in duplicate. T-PER soluble fractions were loaded directly onto plates, whereas the formic acid supernatants (insoluble fractions) were diluted 1:20 in a neutralization buffer (1 mol/L Tris base and 0.5 mol/L NaH₂PO₄) before loading. All samples were loaded in duplicate and incubated overnight at 4°C. Plates were then washed and probed with either horseradish peroxidase-conjugated anti-Aβ₄₀ or anti-Aβ₄₂ (Drs. Vitaly Vasilevko and David H. Cribbs, University of California, Irvine) overnight at 4°C. To develop the plate, the chromogen 3,3′,5,5′-tetramethylbenzidine was added followed, by 30% phosphoric acid to stop the reaction. The plates were read at 450 nm using a plate reader (Molecular Dynamics, Sunnyvale, CA). The readings were then normalized to protein concentrations of the samples.