title: Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides.
abstract: Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.
authors.affiliation: Department of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Grönwall
authors.forename: Andreas
authors.initials: A
authors.lastname: Jonsson
authors.forename: Sara
authors.initials: S
authors.lastname: Lindström
authors.forename: Elin
authors.initials: E
authors.lastname: Gunneriusson
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.forename: Nina
authors.initials: N
authors.lastname: Herne
mesh_terms: D016229:Amyloid beta-Peptides / Q000276:immunology* / Q000378:metabolism*; D000915:Antibody Affinity; D018604:Epitope Mapping; D019151:Peptide Library; D058225:Plaque, Amyloid; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D016229:Amyloid beta-Peptides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins