96-well plates were coated with 100 ng agalsidase-alfa (Takeda) per well over night at 4 °C and washed three times with PBS. For negative controls, 100 ng BSA per well was used. After blocking with 2% BSA/PBS for 1 h at room temperature, wells were washed once with PBS. To detect anti-agalsidase-alfa antibodies from patients’ sera, serial dilutions from individual raw sera were loaded into the wells and incubated for 2 h at room temperature. After five washing steps with 0.1% Tween-20/PBS, anti-hIgG antibodies conjugated with HRP (ab98624, Abcam working concentration: 20 ng/mL) were applied and incubated for 1 h at room temperature. Wells were washed again five times with 0.1% Tween-20/PBS. For IgG detection 50 µL 1-Step TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Darmstadt, Germany) were added to the wells, followed by 50 µL 2 M sulfuric acid to stop the reaction after 15 to 20 min. Absorption was measured at 450 nm. A serial dilution of the anti-alpha-galactosidase A reference antibody (Lenders et al. Int. J. Mol. Sci. 2021, 22, 2680) starting with 800 pg/µL, was used as reference.
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Submitted Aug 04 2021