Longitudinal biomarkers in amyotrophic lateral sclerosis.

Objective: To investigate neurodegenerative and inflammatory biomarkers in people with amyotrophic lateral sclerosis (PALS), evaluate their predictive value for ALS progression rates, and assess their utility as pharmacodynamic biomarkers for monitoring treatment effects.

Methods: De-identified, longitudinal plasma, and cerebrospinal fluid (CSF) samples from PALS (n = 108; 85 with samples from ≥2 visits) and controls without neurological disease (n = 41) were obtained from the Northeast ALS Consortium (NEALS) Biofluid Repository. Seventeen of 108 PALS had familial ALS, of whom 10 had C9orf72 mutations.

HTRF analysis of soluble huntingtin in PHAROS PBMCs.

Objective: We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker.

Methods: Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenine-guanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures.

Optimization of an HTRF Assay for the Detection of Soluble Mutant Huntingtin in Human Buffy Coats: A Potential Biomarker in Blood for Huntington Disease.

A means for measuring levels of soluble huntingtin proteins in clinical samples is essential for assessing the biological effects of potential mutant huntingtin (mtHtt) modifying treatments being developed for Huntington's disease (HD). We have optimized a previously described cell-based Homogeneous Time Resolved Fluorescence method that can measure soluble mtHtt and its ratio to the total Htt (tHtt) in blood buffy coats [1]. The results of the optimization and assay qualification indicate the assay to be specific for mtHtt in HD compared to Control subjects, highly sensitive, and technically and biologically reproducible.

Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count.

Huntington's disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin.