Victor2 fluorometer

The following method was applied for the determination of AGLA (alpha-galactosidase) activity in DBS (dried blood spots). The used protocol implies extraction of the AGLA from DBS, incubation with a synthetic substrate for a defined amount of time, and detection of the enzymatic product using fluorimetry. AGLA enzymes are extracted from dry blood spots in sodium acetate buffer in a 96-well plate at 37°C with agitation. On top of the extracts, a specific synthetic substrate (4-methylumbelliferyl-α-D-galactopyranoside) is added, and the plates are incubated at 37°C for 4–6 h. The reaction is stopped by adding carbonate buffer (changing the pH to 10.7). The quantitation of the product (4-MU) was performed by fluorimetry on a Victor2 Fluorometer (PerkinElmer) using an external calibration line of 4-methylumbelliferone. The results of the enzymatic activity determination were calculated in μmol/L/h. Enzymatic AGLA levels were not analyzed in females as previous studies have shown an inconsistent relationship between normal AGLA levels and GLA mutation status in FD subjects. On the other hand, normal AGLA levels in men predict a normal GLA mutation status, and thus, further Lyso-Gb3 level determination and genetic studies were performed in men only in the case of abnormal AGLA levels.

To investigate the activity of the HN protein, the haemadsorption assay was used. Monolayer cultures of MARC 145 cells in 96-well plates were inoculated with the bat paramyxovirus B16-40 at an MOI of 0.1. After 48 hours, the cells were gently washed with PBS and incubated for 1 hour with 0.5% chicken red blood cells (RBCs) in PBS (pH 7.4) at room temperature. Then, the cells were washed with PBS twice to remove unbound RBCs and observed under a microscope. In addition, the neuraminidase assay was performed with the viral isolate using the NA-Fluor Influenza Neuraminidase Assay Kit (Thermo) according to the manufacturer’s protocol. The virus was diluted 2-fold with the NA-Fluor 2× assay buffer in a black 96-well plate and incubated for 1 hour at 37 °C with the NA-Fluor substrate. After adding the stop solution, the plate was read at the excitation and emission wavelengths of 355 nm and 460 nm, respectively, using a PerkinElmer VICTOR2 fluorometer.