Description
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.
Formats
Our 100 assay point kit allows you to run 100 wells in 96-well format, using a 100 µL reaction volume (10 µL of sample).
Our 500 assay point kit allows you to run 500 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample).
Our 5,000 assay point kit allows you to run 5,000 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample
Features
No-wash steps, no separation steps
ELISA alternative technology
Sensitive detection
Broad sample compatibility
Small sample volume
Results in less than 4 hours
AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
Principle of the AlphaLISA Human & Mouse Mutant HTT assay
The AlphaLISA Mutant HTT assay is based on an AlphaLISA sandwich immunoassay involving a biotinylated anti-target antibody bound to Streptavidin-coated AlphaLISA Donor beads and an anti-target antibody conjugated to AlphaLISA Acceptor beads. One antibody is directed against the total HTT protein, and the second recognizes specifically the mutant polyQ domain. In the presence of the target, both antibodies bind to Mutant HTT, and the beads come into proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer within the Acceptor beads, resulting in emission with λmax at 615 nm. The intensity of the signal is directly proportional to the concentration of Mutant HTT present in the sample (cell lysate or tissue lysate).
Protocol of the AlphaLISA human & mouse Mutant HTT assay
The AlphaLISA Mutant HTT assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples (cell lysates or tissue lysates) or control lysate are dispensed directly into the assay plate for the detection of Mutant HTT by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.
Validation of AlphaLISA Mutant HTT detection kit on HEK293 transfected with different HTT plasmids
Four HTT plasmids were designed with different repeats of glutamine: 23, 48, 73 or 103 repeats were included in the protein sequence.
HEK293 cells were seeded in a 96-well culture-treated plate (25,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were transfected with HTT plasmids. After 48h of incubation, the cells were lysed with 50 µL of AlphaLISA Lysis buffer (1X) supplemented with protease inhibitors for 30 minutes at RT under gentle shaking. 5 µL of each condition were transferred into a 384-well microplate before the sequential addition of AlphaLISA Total HTT and Mutant HTT detection reagents.
As expected, no signal was obtained with samples containing wild-type HTT (<36Q) either non transfected or Q23. The assay is therefore specific for mutant HTT (polyQ tail > 36Q) and the longer the expansion of the polyQ tail, the higher the signal will be even if the total concentration of protein was similar. The level of Total HTT protein remained constant for all the transfected plasmids tested.
Validation on brain tissue samples collected from WT and mutant HTT mouse models
Briefly, a 10% (w/v) brain stem tissue homogenate was prepared using ice-cold 1X AlphaLISA Lysis Buffer (#AL003C) supplemented with protease inhibitors, and the insoluble fraction of the lysate containing putative mutant HTT aggregates was removed by centrifugation at 3500xg for 10 minutes at 4°C, and the supernatants containing soluble mutant HTT were collected for either direct analysis or dispensed into aliquots for storage at ≤-60°C.
The supernatants containing soluble HTT proteins were analyzed using the AlphaLISA Human & Mouse Total HTT Detection Kit and the AlphaLISA Human & Mouse Mutant HTT Detection Kit. To ensure that the detected analyte was assessed at a concentration compatible with the assay’s linear range, the lysates were pre-diluted just before detection in AlphaLISA Lysis Buffer supplemented with protease inhibitors just before detection (1/8 for Total HTT and Mutant assays).
As expected, no signal was obtained with the Mutant HTT assay on samples collected from WT mice, whereas the soluble mutant protein was clearly detected in tissue collected from premanifest zQ175 mice. Finally, the soluble total HTT protein (WT and mutant forms) was measured in all lysates at a similar level in both samples.
