PHAROS PBMCs 中可溶性亨廷顿蛋白的 HTRF 分析。
HTRF analysis of soluble huntingtin in PHAROS PBMCs.
机构信息
From the Massachusetts General Hospital (M.M.-L., R.E.G., J.J.R., H.D.R., S.M., A.B.Y., S.M.H.), MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Charlestown, MA; University of Rochester Medical Center (S.E., D.O.), Department of Biostatistics and Computational Biology, Rochester, NY; Veterans Administration Hospital (W.M.), Bedford, MA; and Program for Regulatory Science & Medicine (I.S.), Georgetown University, Washington, DC.
出版信息
Neurology. 2013 Sep 24;81(13):1134-40. doi: 10.1212/WNL.0b013e3182a55ede. Epub 2013 Aug 21.
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.
RESULTS
From 363 assayed samples, 342 met quality control standards. Levels of mtHtt and mt/tHtt were higher in 114 subjects with expanded CAG repeats (CAG ≥ 37) compared with 228 subjects with nonexpanded CAG repeats (CAG <37) (p < 0.0001). Analysis of relationships to predicted time to onset or to phenoconversion suggested that the HTRF signal could mark changes during the Huntington disease prodrome or after clinical onset.
CONCLUSIONS
The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.
目的
我们使用均相时间分辨荧光（HTRF）测定法测量了 Prospective Huntington At-Risk Observational Study（PHAROS）中 50%携带亨廷顿病突变风险的受试者血液白细胞中的突变型亨廷顿蛋白（mtHtt）和总亨廷顿蛋白（tHtt）水平，以评估其作为生物标志物的潜力。
方法
使用同时测量 mtHtt 和 tHtt 的抗体通过 HTRF 分析同意的 PHAROS 受试者的外周血单核细胞。mtHtt 水平根据胞嘧啶-腺嘌呤-鸟嘌呤重复长度（CAGn）、人口统计学、预测的临床发病时间或已知的临床发病时间以及可用的临床指标进行标准化。
结果
在 363 个测定样本中，342 个符合质量控制标准。与 228 个无扩展 CAG 重复（CAG <37）的受试者相比，114 个扩展 CAG 重复（CAG ≥37）的受试者的 mtHtt 和 mt/tHtt 水平更高（p <0.0001）。对与预测发病时间或表型转换的关系的分析表明，HTRF 信号可能标志着亨廷顿病前驱期或临床发病后的变化。
结论
HTRF 测定法可有效地测量多中心样本中的 mtHtt，并且可能对靶向亨廷顿蛋白的治疗试验有用。
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