Fig. 1
Antibody characterization and ELISA development. a The interaction of Aβ peptides with K11 was analyzed at a Biacore 3000 at 25 °C. Goat anti mouse IgG was immobilized on a CM5 sensor chip, followed by binding of mouse anti-isoD7-Aβ antibody K11. The binding of isoD7-Aβ (1–18) and Aβ (1–18) to K11 was determined by separate injections of 100 nM of each Aβ peptide. b Analysis of different antibodies for specificity in Sandwich ELISAs. 150 ng of Aβ peptides (1—isoD7-Aβ (1–40); 2—Aβ (1–40); 3, 5, 7—isoD7-Aβ (1–30); 4, 6, 8—Aβ (1–30)) was spotted on a nitrocellulose membrane which was blocked in blocking solution for 1 h. Antibodies 4G8, K11, 6E10, and 3D6 were diluted to 1 μg/ml in blocking solution and incubated with the membrane for 1 h. Anti-mouse antibody conjugated to AP was added and incubated for 1 h, followed by 3 × 5 min washing steps and subsequent colorimetric detection of AP activity by addition of substrates BCIP and NBT. c, d Establishment of Sandwich ELISAs for quantification of isoD7-Aβ and total Aβ concentrations. K11 (c) and total Aβ specific antibody 3D6 (d) were diluted to 2 μg/ml and immobilized on microtiter plates overnight at 4 °C. Blocking occurred for 2 h at room temperature. Standard peptides isoD7-Aβ (1–30) and Aβ (1–30) were serially diluted from 150 pg/ml down to 1.6 pg/ml. After an incubation period of 2 h at 4 °C, plates were washed six times with TBS-T. HRP-conjugated anti-Aβ antibody clone 4G8 was added in a final concentration of 1 μg/ml and incubated for 1 h at 4 °C. After washing with TBS-T, a color reaction with TMB was performed and stopped by the addition of 1.2 N H2SO4. The standard curve was calculated from measured absorption at 450/540 nm by a 4-Parameter-Logistic-Fit: y = (A2 + (A1 − A2)/(1 + (x/x0)^p)