title: Enzyme-linked immunosorbent assay-based method to quantify the association of small molecules with aggregated amyloid peptides.
abstract: This paper describes a simple enzyme linked immunosorbent assay (ELISA) protocol for quantifying the binding of small molecules to aggregated β-amyloid (Aβ) peptides. Amyloid-targeting small molecules have attracted wide interest as potential agents for the treatment or diagnosis of neurodegenerative disorders such as Alzheimer's disease. The lack of general methods to evaluate small molecule-amyloid binding interactions, however, has significantly limited the number of amyloid-targeting molecules that have been studied to date. Here, we demonstrate a general method to quantify small molecule-amyloid binding interactions via a modified quantitative ELISA protocol. A key feature of this protocol is the treatment of commercial ELISA plates with an air plasma to help maintain the desired β-sheet content of the aggregated Aβ upon immobilization of these peptides on to the polystyrene surface. We developed an ELISA-based competition assay on these air plasma-treated plates and evaluated the binding of five previously known amyloid-binding small molecules to aggregated Aβ. We show that this general ELISA-based competition assay can be used to quantify small molecule-amyloid binding interactions in the low nanomolar to low micromolar range, which is the typical range of affinities for many amyloid-targeting diagnostic agents under current development. This simple protocol for quantifying the interaction of small molecules with aggregated Aβ peptides overcomes many limitations of previously reported spectroscopic or radioactivity assays and may, therefore, facilitate the screening and evaluation of a more structurally diverse set of amyloid-targeting agents than had previously been possible.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
authors.forename: Christina C
authors.initials: CC
authors.lastname: Capule
authors.forename: Jerry
authors.initials: J
authors.lastname: Yang
mesh_terms: D000544:Alzheimer Disease / Q000175:diagnosis; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D011137:Polystyrenes / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D016229:Amyloid beta-Peptides; D055767:Immobilized Proteins; D011137:Polystyrenes