Background Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (htt) gene and there is no cure available. Currently, there are several animal models that recapitulate the disease phenotype and knockdown of mutant htt in some of these models have exhibited beneficial therapeutic effects. However, there are limited tools to quantify different forms of soluble full-length htt in biological samples in order to monitor the efficacy of the therapeutic molecules.
Aim We aimed to establish bioassays that can detect and selectively quantify the wild-type (WT) and mutant forms of htt in biological samples.
Methods Two-step no wash bioassays were developed to detect soluble forms of full-length htt by using the AlphaLISA® technology. In each assay, a pair of antibody was used to capture either total or mutant htt. Assay performance was tested in HEK293 cells, WT and BACHD brain homogenates. For selective quantification of the WT htt, brain samples from BACHD mice were incubated with antibody-conjugated magnetic beads to deplete mutant htt.
Results The AlphaLISA® platform was efficient at detecting endogenous and overexpressed forms of soluble htt in HEK293 cells and in brain homogenates. The mutant htt assay showed a signal increase proportional to the length of the glutamine repeat. Total and mutant htt assays revealed differential htt expression in various brain regions (cortex, hippocampus, thalamus and cerebellum) of WT and BACHD mouse models. In addition, our results showed that total htt levels in BACHD brain is only about 40%–50% higher than WT mice, suggesting that this mouse model does not exhibit overt overexpression of the protein.
Conclusions Htt AlphaLISA assays demonstrated robust performance, high sensitivity and broad dynamic range. Hence, these assays can be used as a powerful tool to quantify soluble htt variants in experimental studies using both cellular and tissue samples.
- Soluble huntingtin detection
- BACHD mice
- biomarkers for Huntington's disease
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