4.4. Fluorimetric Alpha-Galalactosidase Assay

To assess the performance of the newly developed method, a comparison against a standard method for human α-galactosidase quantification by fluorimeter was conducted. α-Galactosidase activity was measured by a fluorimetric enzyme assay on 12 sachets from the same batch. The assay mix included 50 μL of raw material corresponding to 500 ng of 4-methylumbelliferyl-α-D-galactopyranoside as a substrate (Sigma Chemical; final concentration of 6.7 mM) and sodium acetate buffer (pH 4.5; final concentration 0.13 M) in a final volume of 300 μL. The reaction mix was incubated for 30 min at 37 °C in the dark. To stop the reaction, 1700 µL of buffer carbonate (0.5 M pH 10.7) was added. The assay was repeated with and without the inclusion of N-acetylgalactosamine (GalNAc) at a 100-mM final concentration in the previous assay mixture. GalNAc is an inhibitor of the lysosomal enzyme α-N-acetylgalactosaminidase. also known as α-galactosidase B because in vitro it has some nonspecific activity toward the artificial substrate used for assay of α-galactosidase B. In this case, substrate solution (no enzyme), blank (only water) and enzyme blanks (enzyme but no substrate) were included for background and non-specific corrections.

Readings were performed to measure fluorescence intensity (Excitation/Emission= 360/445 nm) at room temperature using an end-point setting. A calibration curve was obtained by linear regression of the absorbance readings versus concentration using 10 different solutions with known concentrations of the analyte.