HTRF Human and Mouse HTT Aggregation Kit, 10,000 Assay Points

Protocol of the HTRF Human & Mouse Aggregated HTT assay

The HTRF Aggregated HTT assay can be run in a 96- or 384-well low volume white detection plate (20 µL final). As described here, samples (cell/tissue lysates) are dispensed directly into the assay plate for the detection of HTT aggregates by HTRF reagents. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Analysis of brain tissue samples collected from WT and mutant HTT mouse models

The HTRF Aggregated HTT assay was validated on brain tissues collected from premanifest zQ175 mice and wild-type (WT) littermates, the most extensively studied preclinical knock-in mouse model used for Huntington’s Disease (Landles C. et al. Brain Commun. 2021; 3(1):fcaa231; Smith EJ. et al. Brain Commun. 2023; 5(1):fcad010; Landles et al., Brain Commun. 2024; 6(6):fcae410).

The hippocampus tissues of WT and mutant mice aged 2 or 12 months were lysed following the procedure given in the kit’s package insert. Briefly, a 10% (w/v) tissue homogenate was prepared using ice-cold 1X lysis buffer #2 supplemented with protease inhibitors. The lysates, containing aggregates and soluble HTT proteins, were analyzed using the HTRF Total HTT assay (Cat# 64HTTTPEG/H) assay, the HTRF Mutant HTT assay (Cat# 64HTTMPEG/H), and the HTRF Aggregated HTT assay. To ensure that the detected analyte was assessed at a concentration compatible with the assay’s linear range, the lysates were pre-diluted in 1X lysis buffer #2 supplemented with protease inhibitors just before detection (1/4 for Total HTT assay, 1/8 for Mutant HTT assay, and 1/2 for Aggregated HTT assay).

Results show the soluble Total HTT protein (WT and mutant forms) was measured in all lysates (green bars), and its levels were similar for all samples. No signal was obtained with the Mutant HTT assay on samples collected from WT mice, whereas the soluble mutant protein was clearly detected in hippocampus collected from premanifest zQ175 mice (purple bars). The level of soluble mutant HTT decreased in the 12-month-old mice because of mutant protein aggregation. This aggregation was well detected (blue bars), in agreement with literature.