Development and validation of a SIMOA-based mutant ataxin-3 immunoassay for biomarker studies in SCA3

01/03/2020 to 30/06/2021

The search for molecular marker which are representing disease stages and/ or therapeutic response in neurodegenerative diseases is increasing since years. The development of such marker is highly dependent from high throughput techniques which are screening CSF and peripheral blood for changes during disease progression as well as techniques which allow fast, sensitive and quantitative measurement of the identified targets. Such target for ongoing neurodegenerative processes was identified in several neurodegenerative diseases in the last months, namely neurofilament light chain (Nfl). Here using the highly sensitive and quantitative technique Simoa which is based on two protein specific antibodies labelled with capture and detector fluorophore demonstrated a close correlation of CSF Nfl levels with the Nfl levels in the peripheral blood. The detected protein amounts of around 70 pg/ml Nfl in SCA3 patients and 20 pg/ml in controls in plasma (Wilke et al., 2018) make clear why the development and validation of highly sensitive techniques are so important. Earlier we developed a TR-FRET-based immunoassay to measure mutant ataxin-3 in the brain and isolated human mononuclear cells (PBMCs). But in the end, the TR-FRET assay was not sensitive enough to measure mutant ataxin-3 in plasma/ serum or CSF. Therefore, in a second step we developed a high specific and sensitive immunoassay on SMC platform and demonstrated amounts of around 10 pg/ml mutant ataxin-3 in CSF and up to 200 pg/ml mutant ataxin-3 in plasma of SCA3 patients. Summarizing the information above demonstrated that there are highly sensitive techniques to measure pg amounts of different proteins, but all the techniques are cost intensive and mostly dependent from the pipetting researcher. Therefore, combining the different proteins, here mutant ataxin-3 and Nfl, on one platform would allow to measure both proteins in one biosample, which is reducing the amount of blood take per visit from each patient, and also can reduce costs and time. Therefore, our aim in this application would be to use our knowledge from developing and validating TR-FRET and SMC technology and adapt the mutant ataxin-3 measurement on the Simoa-based technology. This will help us in later clinical studies to measure both molecular markers, Nfl and mutant ataxin-3, on one platform, in one biosample with the already widely used and fully automated system Simoa.


As nearly all done therapeutic studies failed in the last years because of missing sensitive primary outcome parameters, we hopefully pave the way with our study for new successful therapeutic studies in the field of Spinocerebellar Ataxia Type 3. Thereby, the combination of the disease protein itself (ataxin-3), which is increasing with disease severity and the target for disease protein lowering therapies together with the neurodegeneration marker Nfl can lead to robust outcome parameter for lowering disease protein expression and lowering neurodegenerative processes in the brain.