Validation of high-sensitivity 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 () 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 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 and 0.20 nmol/mL/3 h, 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.