Polyglutamine (polyGln) aggregates are neuropathological markers of expande
d CAG repeat disorders, and may also play a critical role in the developmen
t of these diseases. We have established a highly sensitive, fast, reproduc
ible, and specific assay capable of monitoring aggregate-dependent depositi
on of polyglutamine peptides. This assay allows detailed studies on various
aspects of aggregation kinetics, and also makes possible the detection and
quantitation of low levels of "extension-competent" aggregates. In the sim
plest form of this assay, polyGln aggregates are made from chemically synth
esized peptides and immobilized onto microplate wells. These wells are incu
bated for different times with low concentrations of a soluble biotinylated
polyGln peptide. Europium-streptavidin complexation of the immobilized bio
tin, followed by time-resolved fluorescence detection of the deposited euro
pium, allows us to calculate the rate (fmol/h) of incorporation of polyGln
peptides into polyGln aggregates. This assay will make possible basic studi
es on the assembly mechanism of polyGln aggregates and on critical features
of the reaction, such as polyGln length dependence. The assay also will be
a valuable tool for screening and characterizing antiaggregation inhibitor
s. It will also be useful for detection and quantitation of aggregation-com
petent polyGln aggregates in biological materials, which may prove to be of
critical importance in understanding the disease mechanism. (C) 2001 Acade
mic Press.