Mutant HTT assay principle

The Mutant HTT assay is based on a TR-FRET sandwich immunoassay involving two specific antibodies, one labelled with Tb3+ cryptate (donor) and the other with d2 (acceptor). One antibody is directed against the N-terminal part of the protein, and the second recognizes specifically the mutant polyQ domain. Both antibodies bind to soluble Mutant HTT, and the donor-acceptor proximity enables a fluorescent TR-FRET signal. The intensity of the signal is directly proportional to the concentration of soluble Mutant HTT present in the sample (cell lysate or tissue lysate).

Mutant HTT assay protocol

The Mutant 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) or standards are dispensed directly into the assay plate for detection of Mutant HTT 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.

Mutant HTT standard curve

The recombinant proteins WT mouse HTT-Q7 (Coriell Institute, #CH02324), mutant human HTT-Q48 (Coriell Institute, #CH02580) and mutant human HTT-Q73 (Coriell Institute, #CH02581) were purchased separately. They were serially diluted in lysis buffer #2 (1X), following the procedure given in the kit’s package insert. The HTRF signal, expressed as a delta ratio, was plotted as a function of the HTT concentrations tested.

As expected, the Mutant HTT assay is able to measure mutant HTT proteins containing different sizes of polyQ (48Q & 73Q tested here), while it does not recognize the WT form of the protein.

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 on the Mutant HTT levels for the three different brain tissues.