TTR fibril inhibition assay

A stock solution of TTR-V122I (approximately 5 mg/mL) was diluted into buffer with final concentrations of 0.2 mg/mL TTR, 30 mM sodium acetate, 5 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, 0.02% sodium azide, and varying concentrations of mAb at pH 7.2. This sample was dialyzed against the same buffer at pH 4.5 for 3 h at room temperature. Samples were then incubated at 37 °C for 72 h. The extent of fibril formation was probed by Thioflavin T (ThT) (Sigma-Aldrich, St Louis, MO). A five-fold molar excess of ThT was added to each sample and left at room temperature for 30 min before measurements were taken. ThT fluorescence was monitored using a Photon Technology International C60 spectrofluorometer. The photomultiplier gain was varied and excitation and emission slit widths set to 2–4 nm to maximize signal to noise. Fluorescence measurements were made using 430 and 480 nm as excitation and emission wavelengths, respectively.

The ThT fluorescence was normalized with the following formula to enable comparison of fluorescence measurements taken on different days: