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 and mouse HTT assay
The AlphaLISA Huntingtin (HTT) assay is based on an AlphaLISA sandwich immunoassay involving a biotinylated anti-HTT antibody bound to Streptavidin-coated AlphaLISA Donor beads and an anti-HTT antibody conjugated to AlphaLISA Acceptor beads. Both antibodies are directed against the HTT protein. In the presence of the HTT, both antibodies bind to 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 HTT present in the sample (cell cell lysate or tissue lysate).
Protocol of the AlphaLISA human & mouse Total HTT assay
The AlphaLISA Total 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 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 Total HTT detection kit 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 until 80% confluence . After a 48h incubation, the cells were lysed with 3 mL of AlphaLISA Lysis Buffer (1X) supplemented with proteases inhibitors for 30 minutes at RT under gentle shaking.
After centrifugation of the cell lysates, the supernatants were serially diluted in supplemented AlphaLISA Lysis Buffer (1X), and 5 µL of each dilution were transferred into a 384-well microplate before the sequential addition of AlphaLISA Total HTT detection reagents.
The AlphaLISA 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.
Validation of AlphaLISA Total 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, the total HTT assay showed a constant level of HTT protein for all the plasmids transfected, with minimal signal from the untransfected HEK293 cells showing a good efficacy of transfection. For the Mutant HTT assay, no signal was obtained with samples containing wild-type HTT (<36Q) either non transfected or Q23.
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 are collected for 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 with a similar level.
