Biotin-avidin interaction-based screening assay for Alzheimer's beta-peptide oligomer inhibitors.
In vitro testing for inhibitors of oligomer formation of pathologically misfolded proteins such as Alzheimer's beta-peptide (Abeta) has been limited by the lack of a suitably sensitive high-throughput method for measuring oligomers. Even with the development of oligomer-specific antibodies and a single-site antibody assay, there are multiple controls required to rule out false positives due to compound interactions with the epitopes on the peptide that are recognized by the antibodies or with the antibodies themselves, and the immunoreagents are expensive. A non-radioactive non-immunological method for the measurement of subnanomolar concentrations of Alzheimer's beta-peptide residues 1-42 [Abeta(1-42)] oligomers incorporating the biotin-avidin interaction that has been a workhorse for screening assays is applied here in a single-site NeutrAvidin capture/labeled streptavidin detection configuration to specifically recognize multimeric (>20kDa) oligomers of N-alpha-biotinyl-Abeta(1-42) (bio-Abeta42) but not monomeric bio-Abeta42. The high affinity and specificity of the biotin interaction with NeutrAvidin and streptavidin obviate interference by non-biotin-containing compounds. The reagents are inexpensive and can be applied to any misfolding/oligomerizing peptide or protein that can be biotinylated at a single site.
LeVine
Harry
H
Department of Molecular and Cellular Biochemistry, Chandler School of Medicine, and Center on Aging, University of Kentucky, Lexington, KY 40536, USA. hlevine@email.uky.edu
D000544:Alzheimer Disease / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008956:Models, Chemical; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42); D001360:Avidin; D001710:Biotin