MABN687 Sigma-AldrichAnti-β-amyloid fibril-specific, clone B10, AP Antibody

Oligomers are intermediates of the β-amyloid (Aβ) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer's disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aβ conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aβ oligomers, such as those formed by Aβ (1-40) and Aβ (1-42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aβ residues 18-20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aβ assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aβ fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aβ conformers.

Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1-40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10-fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils.

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimer's disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of beta-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Abeta amyloid fibrils from disaggregated Abeta peptide as well as from specific Abeta oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Abeta protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation.