Example 8
This example provides an alternative in vitro activity assay for SGSH-Fc fusion proteins. The assay is adapted from Karpova et al., J. Inherit. Metab. Dis., 19:278-285 (1996).
The standard reaction mixtures consisted of 10-15 μg of protein and 20 μL MU-α-GlcNS (5 or 10 mmol/L, respectively) in Michaelis' barbital sodium acetate buffer, pH 6.5 (29 mmol/L sodium barbital, 29 mmol/L sodium acetate, 0.68% (w/v) NaCl, 0.02% (w/v) sodium azide; adjusted to pH 6.5 with HCl) and the reaction mixtures were incubated for 17 h at 37° C. MU-α-GcNS is available from Moscerdam Substrates. After the first incubation, 6 μl twice-concentrated McIlvain's phosphate/citrate buffer, pH 6.7, containing 0.02% sodium azide and 10 μl (0.1 U) yeast a-glucosidase (Sigma) in water were added and a second incubation of 24 h at 37° C. was carried out. Long incubations at 37° C. (17-24 h) were carried out in 96-well plates which were sealed airtight with broad sticky tape, limiting evaporation to <15%. Next, 200 μL 0.5 mol/L Na2CO3/NaHCO3, pH 10.7, was added, and the fluorescence of the released 4-methylumbelliferone (MU) was measured on a Fluoroskan (Titertek) fluorimeter. Protein was determined as described previously (van Diggelen et al., Clin. Chim. Acta., 187:131-139 (1990)).
