验证高灵敏度荧光测定法,以定量检测 GM1-神经节苷脂病患者脑脊液和血清中的β-半乳糖苷酶活性

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
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引用次数: 0

摘要

GM1-神经节苷脂病(GM1)是一种溶酶体贮积症,由β-半乳糖苷酶1基因(GLB1)突变引起,导致β-半乳糖苷酶(β-gal)活性降低。这种酶的缺乏会导致神经元变性、发育迟缓和早期死亡。开发疾病调节疗法需要一种灵敏的β-gal酶活性测定方法。我们优化了荧光测定法,用于定量分析人脑脊液(CSF)和血清中的β-gal活性,以开发GLB1基因替代疗法。通过评估灵敏度、精确度、准确性、平行性、特异性和样品稳定性,对测定分析性能进行了鉴定。CSF和血清β-gal活性测定的灵敏度分别为0.05 nmol/mL/3hr和0.20 nmol/mL/3hr。人CSF和血清的测定精度(以测定间变异系数百分比表示)分别为15%和20%。研究还考察了分析前因素对β-gal活性的影响,样品采集后的快速处理和冷冻对保持酶的活性至关重要。通过这些检测方法,可以测量健康人和 GM1-神经节苷脂病患者的脑脊液和血清中的β-gal 活性。这种脑脊液β-gal活性测定法是同类测定中首个灵敏度足以定量测定GM1患者脑脊液样本中β-gal酶活性的方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Validation of high-sensitivity fluorometric assays to quantitate cerebrospinal fluid and serum β-galactosidase activity in patients with GM1-gangliosidosis

Validation of high-sensitivity fluorometric assays to quantitate cerebrospinal fluid and serum β-galactosidase activity in patients with GM1-gangliosidosis

GM1-gangliosidosis (GM1) is a lysosomal storage disorder caused by mutations in the galactosidase beta 1 gene (GLB1) that leads to reduced β-galactosidase (β-gal) activity. This enzyme deficiency results in neuronal degeneration, developmental delay, and early death. A sensitive assay for the measurement of β-gal enzyme activity is required for the development of disease-modifying therapies. We have optimized fluorometric assays for quantitative analysis of β-gal activity in human cerebrospinal fluid (CSF) and serum for the development of a GLB1 gene replacement therapy. Assay analytical performance was characterized by assessing sensitivity, precision, accuracy, parallelism, specificity, and sample stability. Sensitivity of the CSF and serum β-gal activity assays were 0.05 nmol/mL/3hr and 0.20 nmol/mL/3hr, respectively. Assay precision represented by inter-assay percent coefficient of variation of the human CSF and serum was <15% and <20%, respectively. The effect of pre-analytical factors on β-gal activity was examined, and rapid processing and freezing of samples post-collection was critical to preserve enzyme activity. These assays enabled measurement of CSF and serum β-gal activities in both healthy individuals and patients with GM1-gangliosidosis. This CSF β-gal activity assay is the first of its kind with sufficient sensitivity to quantitatively measure β-gal enzyme activity in CSF samples from GM1 patients.

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来源期刊
Molecular Therapy-Methods & Clinical Development
Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.90
自引率
4.30%
发文量
163
审稿时长
12 weeks
期刊介绍: The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.
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