Fig. 1

Assessment of antibody selectivity. A–D A series of synthetic N-terminal amyloid-β (Aβ) variants was separated by isoelectric focusing in microcapillaries, immobilized photochemically to the inner capillary wall and probed with the indicated monoclonal anti-Aβ antibodies. The peptides were loaded as a mixture or individually, as indicated. A Monoclonal antibody (mAb) 6E10 recognized Aβ40 variants starting at Asp(1), Ala(2), Glu(3) pyro-Glu(3), Phe(4), Arg(5) and Val(−3). Aβ11–40 was not detected. B MAb 3D6 strongly detected Aβ1–40, but essentially none of the other tested N-terminal Aβ variants. C MAb 4G8 recognized all of the tested Aβ variants. D MAb 1E8 recognized Aβ1–40, Aβ2–40, Aβ3–40 and AβN3pE–40. In addition, the N-terminally elongated Aβ−3–40 was recognized, albeit with a comparatively small signal. Aβ4–40, Aβ5–40 and Aβ11–40 were not detected. E MALDI-TOF-MS mass spectrum of Aβ peptide variants immunoprecipitated by monoclonal antibody 1E8. A mixture of synthetic Aβ peptides and Aβ related peptides was subjected to magnetic bead immunoprecipitation with mAb 1E8 followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in reflector mode. The starting material included synthetic peptides corresponding to Aβ1–38 (calculated monoisotopic mass [M + H]⁺_{mono, calc} = 4130.019), Aβ1–40 (4328.156), Aβ1–42 (4512.277), Aβ2–40 (4213.129), Aβ3–40 (4142.092), AβN3pE–40 (4124.081), Aβ4–40 (4013.049), Aβ5–40 (3865.981), Aβ11–40 (3150.677), Aβ−3–40 (APP669–711; 4686.360) and the model peptide Aβ−23–16 (APP649–687; 4396.174). Under the experimental conditions, mAb 1E8 immunoprecipitated Aβ1–38, Aβ1–40, Aβ1–42, Aβ2–40, Aβ3–40 and the N-terminally elongated Aβ-related peptides Aβ−3–40 and Aβ−23–16 as indicated by the observed monoisotopic masses annotated in the mass spectrum. Aβ, amyloid-β; AβN3pE–40, Aβ40 peptide carrying an N-terminal cyclized pyroglutamic acid residue [pyro Glu(3)] in position 3 of the canonical Aβ amino acid sequence