High throughput monitoring of amyloid-β(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-beta (A beta)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic A beta(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of A beta(42). Modeling suggests that oligomerized A beta(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in A beta(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in A beta(42) oligomerization. Using ELISA and AlphaLISA, we found that A beta(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1nM A beta(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the A beta(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit A beta(42) oligomerization.