Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay

Abstract

Background: The use of biomarkers has become a major component of clinical trial design. In Huntington’s disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein. Objective: We aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples. Methods: An ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples. Results: A novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF. Conclusion: We have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington’s Disease

“…Samples were analyzed in technical triplicates, using 2, 5, and 2 ug/well of protein lysate for cortex, quadriceps, and liver tissues samples, respectively. PBMC and plasma samples were assayed for mHTT quantification by the 2B7-MW1 HTT SMC assay (2B7/MW1), as similarly described in previous work . The analysis of each sample was carried out in triplicate, using 50 ng of total protein/well of PBMC lysates, or using 1 μL of plasma/well.…”

“…HRMS (TOF) calcd for C 19 H 22 FN 7 O [M + H] + 384.1948384. , found 384.1973pyrrolidin-1-yl)-N- (8-fluoro-2-(fluoromethyl)imidazo [1,2-a]pyridin-6-yl)pyrazine-2-carboxamide (42). (3R)-N-cyclopropylpyrrolidin-3-aminedihydrochloride (see Supporting Information, 985 mg, 4.95 mmol), methyl 5-chloro-2pyrazinecarboxylate (93, 854 mg, 4.95 mmol), 1,4-dioxane (100 mL), and N,N-diisopropylethylamine (2 mL, 11.5 mmol) were combined and heated to 100 °C for 3 days.…”

“…PBMC and plasma samples were assayed for mHTT quantification by the 2B7-MW1 HTT SMC assay (2B7/MW1), as similarly described in previous work. 42 The analysis of each sample was carried out in triplicate, using 50 ng of total protein/well of PBMC lysates, or using 1 μL of plasma/well.…”

Salivary Huntingtin protein is uniquely associated with clinical features of Huntington’s Disease

“…Total Htt and mHtt was measured in each bio uid using the 2B7-D7F7 Single Molecule Counting (SMC) assay and three technical replicates [21] for tHtt, mHtt was measured using the 2B7-MW1 SMC assay and two technical replicatesHtt [22] , on an Erenna® platform. The 2B7-MW1 SMC assay is speci c for mHtt, whereas the 2B7-D7F7 assay quanti es Htt protein in a polyglutamine length-independent manner (mHtt and non-expanded wild-type Htt combined) [21,22] . Plasma samples were analyzed using 15μL/well for 2B7-D7F7 SMC assay and 135μL/well for the 2B7-MW1 SMC assay.…”

Salivary Huntingtin protein is uniquely associated with clinical features of Huntington’s disease

“…Total HTT and mHTT was measured in each biofluid using the 2B7-D7F7 Single Molecule Counting (SMC) assay and three technical replicates 21 for tHTT, mHTT was measured using the 2B7-MW1 SMC assay and two technical replicatesHTT 22 , on an Erenna ® platform. The 2B7-MW1 SMC assay is specific for mHTT, whereas the 2B7-D7F7 assay quantifies HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild-type HTT combined) 21,22 . Plasma samples were analyzed using 15 μL/ well for 2B7-D7F7 SMC assay and 135 μL/well for the 2B7-MW1 SMC assay.…”