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HTRF Human/Mouse Total Huntingtin (HTT) Detection Kit HTRF®

The HTRF Total Huntingtin kit is designed for the simple and rapid quantification of soluble total HTT (WT & mutant forms) in cell/tissue lysates.

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  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • Highly accurate Highly accurate

The HTRF Total Huntingtin kit is designed for the simple and rapid quantification of soluble total HTT (WT & mutant forms) in cell/tissue lysates.

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Overview

Huntingtin (HTT) is a cytoplasmic protein which is highly expressed in the brain, and whose anti-apoptotic role is critical for neuronal survival. 

The wild-type (WT) protein has a functional structure, with a "normal" polyglutamine (polyQ) domain containing less than 36Q. The mutant HTT protein harbors an abnormally long polyQ tract (> 36Q), which causes the aggregation of the no longer functional protein. HTT aggregation leads to the selective neuronal cell death responsible for Huntington's Disease.   

Benefits

  • SPECIFICITY
  • PRECISION

Total HTT assay principle

The Total 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). Both antibodies bind to soluble Total HTT (WT & mutant forms), and the donor-acceptor proximity enables a fluorescent TR-FRET signal. The intensity of the signal is directly proportional to the concentration of soluble Total HTT present in the sample (cell lysate or tissue lysate).

Principle of the HTRF total HTT assay

Total HTT assay protocol

The Total 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 the detection of Total 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. 
Assay protocol of the HTRF total HTT kit

Technical specifications of Total HTT kit

Sample size10 µL
Final assay volume20 µL
Time to resultsOvernight at RT
Kit componentsFrozen control lysate, detection antibodies, buffers & protocol
LOD & LOQ (in Lysis Buffer #2)1.6 ng/mL & 6.3 ng/mL
Assay range6.3 - 750 ng/mL
Species cross-reactivityHuman, mouse, & rat
Assay specificitySoluble wild-type & mutant HTT forms

Total HTT standard curve

The recombinant proteins WT mouse HTT-Q7 (Coriell Institute, #CH02324), WT human HTT-Q23 (Coriell Institute, #CH02578), 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 Total HTT assay is able to measure all WT and mutant forms of HTT protein.

Total HTT assay on recombinant HTT proteins

Total HTT assay precision

Intra-assay (n=24)

Sample[WT human HTT-Q23] (ng/mL)CV
123.49%
293.85%
33757%

Mean CV7%

The 3 samples correspond to 3 different concentrations (low, medium, and high) of recombinant WT human HTT-Q23 protein diluted in lysis buffer #2 (1X). Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.


Inter-assay (n=3)

Sample[WT human HTT-Q23] (ng/mL)CV
123.42%
293.80.4%
33751%

Mean CV1.1%

The 3 samples correspond to 3 different concentrations (low, medium, and high) of recombinant WT human HTT-Q23 protein diluted in lysis buffer #2 (1X). Each of the 3 samples was measured in 3 different experiments, and the % CV was calculated for each sample.

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.

HTRF Mutant HTT, Total HTT and GAPDH assays on WT and mutant mouse cortex lysates
HTRF Mutant HTT, Total HTT and GAPDH assays on WT and mutant mouse cerebellum lysates

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.

Comparison HTRF vs. WB on Mutant and Total HTT assays in mouse 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.

Total HTT assay on SH-SY5Y cell lysate dilutions
Total HTT assay on Neuro-2a cell lysate dilutions

Product Insert HTT Total Kit / 64HTTTPEG-64HTTTPEH

64HTTTPEG-64HTTTPEH - Product Insert

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