Figure 2. Affinity analysis of Aβ-specific Anticalins by ELISA.

Binding activity was analysed in a capture ELISA for the biotinylated full-length Aβ40 (A, B) and MBP-Aβ40 (C) as well as for the short biotinylated peptides Aβ1–11 and Aβ16–27 (D). The purified Anticalins were immobilized on to microtitre plates via the Strep-tag II specific antibody StrepMAB-Immo and incubated with a dilution series of the biotinylated Aβ targets (filled symbols) or control proteins (empty symbols), i.e. ovalbumin (Ova) and the MBP. Bound targets were subsequently detected with an ExtrAvidin-Alkaline Phosphatase conjugate, followed by a chromogenic reaction. All Anticalins showed specific binding of the full-length Aβ targets in the low nanomolar range (for the Trx-Aβ28 target cf. Supplementary Figure S3). In addition, there was detectable binding activity towards the central peptide Aβ16–27, but not towards the N-terminal peptide Aβ1–11, as shown here for S1A4 and US7.