To determine the IgG isotype of neutralizing anti-drug antibodies, ELISAs were performed as follows. 96-well Maxisorp ELISA plates (Thermo Scientific) were coated overnight at 4°C with 50 ng agalsidase-alfa, washed 3x with PBS, and blocked for 2h at RT with 100 µl PBS/BSA 2% (per well). Subsequently, wells were washed 5x with PBS/Tween 0.1%. Purified total IgGs from patients) were used as primary antibodies and were applied in serial dilutions (1:250 1:500 1:1,000) in PBS/BSA 2% overnight at 4°C. Next day, cells were washed again 5x PBS/Tween 0.1% and incubated for 1 h at RT with IgG isotype-specific anti-human IgG-HRP (mouse α-human IgG isotype 1 [ab99774] mouse α-human IgG isotype 2 [ab99779] rabbit α-human IgG isotype 3 [ab86253] mouse α-human IgG isotype 4 [ab99823] goat α-human IgA [ab97215] mouse α-human IgE [ab99806]). Rabbit α-human IgG isotype 3 was detected with goat α-rabbit IgG-HRP [sc-2054, Santa Cruz Biotechnology,] as secondary antibody (diluted 1:5,000). After 5 additional washes, 100 µl 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific) was added to each well, incubated for 10 min, and stopped with 2M sulfuric acid. The absorption was measured at 450 nm and ratios were calculated between enzyme replacement therapy-naïve and -treated status.
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Submitted Sep 11 2018
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