Gas914

GAS914 (Novartis Pharma AG, Basel, Switzerland) is a poly-L-lysine backbone with an average degree of polymerization of 1,000 L-lysines and with 23-28% of lysines derivatized with αGal (27 (link)).

Anti-Gal IgM and IgG antibodies were determined by ELISA, as previously described [30 (link)]. Furthermore, we also developed an in-house method for determining xenoantibody reactivity, anti-Gal and -non-Gal antibodies, by flow cytometry. Briefly, confluent PAEC were harvested with TripLE Express (Thermo Fisher Scientific, Waltham, MA, USA), washed, and used to detect possible carbohydrate reactivities. To this end, mouse sera diluted at 1% or 0.5% in PBS 1% bovine serum albumin (Merck) were incubated with PAEC alone or with saturating concentrations of GAS914 (0.5 mg/mL, Novartis, Basel, Switzerland) for 30 min at 4 °C. GAS914 binds anti-Gal antibodies and blocks their reactivity [32 (link)]. IgM and IgG reactivities were measured using the secondary antibodies goat anti-mouse IgM Alexa fluor 647 (1/200 dilution) and goat anti-mouse IgG PE (1/150 dilution) (both from Thermo Fisher Scientific) and a Gallios flow cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA). The mean fluorescence intensity (MFI) was determined (for each individual and time point) after subtracting the background established with the secondary antibody alone or with sera with GAS914. The reactivity remaining following GAS914 competition was considered to be associated with anti-non-Gal antibodies.