Technical specifications of Total HTT kit

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

The HTRF Total 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). The cortex and cerebellum tissues of three WT mice (#1, #2, & #3) and three mutant zQ175 mice (#4, #5, & #6) were lysed following the procedure given in the kit’s package insert. Briefly, a 5% (w/v) tissue homogenate was prepared using ice-cold 1X lysis buffer #2 supplemented with protease inhibitors, and the insoluble fraction of the lysate containing putative mutant HTT aggregates was removed by centrifugation. The supernatants containing soluble HTT proteins were analysed using the HTRF Total HTT assay and the HTRF Mutant HTT assay (Cat# 64HTTMPEG/H). To check that the protein extraction was similar for each sample, the level of the housekeeping protein GAPDH was also measured using the HTRF GAPDH assay (Cat# 64GAPDHPEG/H). 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 1/100 for GAPDH assay).

The levels of GAPDH were similar for all samples (green bars), demonstrating that the tissue lysates contained comparable total protein contents. No signal was obtained with the Mutant HTT assay on samples collected from WT mice, whereas the soluble mutant protein was clearly detected in cortex and cerebellum tissues collected from premanifest zQ175 mice (blue bars). As expected, the soluble total HTT protein (WT and mutant forms) was measured in all lysates (red bars), and its levels correlate well with the small variations observed for the Mutant HTT protein levels.

 

 

Side-by-side comparison of HTRF and Western Blot for the analysis of Mutant and Total HTT in brain tissues

A side-by-side comparison of HTRF Mutant & Total HTT assays and the Western Blot technique was performed on several brain tissues (cortex, hippocampus, and cerebellum) collected from one WT mouse and one premanifest zQ175 mouse. Tissue lysates were prepared as described in the previous section and the supernatants containing soluble HTT proteins were analysed, either by HTRF using the HTRF Mutant & Total HTT assays, or by Western Blot. To ensure that the detected analyte was assessed at a concentration compatible with the assay’s linear range for each detection method, the lysates were pre-diluted in 1X lysis buffer #2 supplemented with protease inhibitors just before detection (for HTRF: 1/8 for Mutant HTT assay and 1/4 for Total HTT assay; for Western Blot: 1/20 for both assays).

The results obtained using HTRF Mutant & Total HTT assays are comparable with those obtained by Western Blot. With both methods, no soluble mutant HTT was detected in brain tissues collected from WT mice, whereas the protein was properly measured in all samples collected from premanifest zQ175 mice. As expected, the soluble total HTT protein (WT and mutant forms) was measured in all lysates, and its levels correlate well with the differences observed for the Mutant HTT levels among the three different brain tissues.

 

Validation of Total HTT assay on human and mouse neuroblastoma cell lines

Human SH-SY5Y and mouse Neuro-2a neuroblastoma cell lines were cultured in T175 flasks in complete culture medium at 37°C-5% CO2. After a 48h incubation, the cells were lysed with 3 mL of lysis buffer #2 (1X) for 30 minutes at RT under gentle shaking. After centrifugation of the cell lysates, the supernatants were serially diluted in lysis buffer #2 (1X), and 10 µL of each dilution were transferred into a low volume white microplate before the addition of 10 µL of HTRF Total HTT detection reagents.

The HTRF Total HTT assay was suitable for the measurement of the levels of soluble WT human and mouse HTT proteins present in SH-SY5Y and Neuro-2a cell lysates.