AlphaLISA Human and Mouse Total HTT Detection Kit, 500 Assay Points

Human & mouse total HTT assay details

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.