Development of an ELISA assay for the quantification of soluble huntingtin in human blood cells

Abstract

BackgroundHuntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.ResultsAn ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.ConclusionsThe method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.

Salivary biomarkers for the diagnosis and monitoring of neurological diseases

“…Blood and CSF Htt protein levels remain fairly low, with a minimum requirement of a 50 ml blood sample to successfully detect Htt protein. In addition to variations in Htt protein concentrations with different blood cell types [165] , [166] , [167] . In a study conducted by Bloom et al.…”

An exploratory double‐blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease

“…The neuropsychiatric battery included the EHDN Problem Behaviours Assessmentshort version (PBA-s) [15], the Hospital Anxiety and Depression Scale [16] combined with the Snaith Irritability Scale (HADS-SIS) [17] and a modified version of the Frontal Systems Behavioural Scale, which was completed by the participants (FrSBe-p) [18]. This modified version of the FrSBe contains 24 items to assess the frequency of certain behaviours and the level of distress caused by those behaviours, but in contrast to the original version we did not use reverse coding for several questions (items [19][20][21][22][23][24], since the reverse coding was confusing for HD patients in a previous study [18]. The cognitive battery included trail making tests A and B [19], symbol digit modalities test (SDMT) [20], Stroop test [21] with evaluation of the total correct answers and the Pittsburgh sleep quality index (PSQI) [22].…”

“…Levels of circulating soluble HTT were analyzed using a MesoScale ELISA method derived from the original ELISA assay with minor modifications [23]. The six time point samples available for 54 patients enrolled in the study were assayed for soluble HTT evaluation.…”

“…The method, described in Massai et al [23], was capable of detecting the HTT protein extracted from PBMC in a reproducible way, and the assay showed a dynamic range of five orders of magnitude, with more than 100-fold signal-to-background ratio. However, statistical analysis of the levels of soluble HTT normalized for total protein did not show statistically significant changes between the treatment arms at any time point ( Figure 1A).…”

Recent Trends in Detection of Huntingtin and Preclinical Models of Huntington’s Disease

“…A recent study reported detection of variations of HTT levels in blood samples using an ELISA method. In addition, the ELISA method could differentiate between peripheral cells isolated from healthy volunteers and HD patients at different disease stages [ 11 ].…”