Anti biotin igg1

Anti-PEG IgG1 (CH2074 except excess biotin experiment, which used CH2076, Silver Lake Research) and anti-PEG IgM (AGP4, IBMS) were used as test Ab and anti-Biotin IgG₁ (Z021, ThermoFisher) and anti-Vancomycin IgM (2F10, Santa Cruz) were used as control Ab. All native Ab were used as is provided by the manufacturer. To prepare deglycosylated Ab, N-glycans on anti-PEG IgG and IgM were removed with rapid non-reducing PNGase F enzyme (New England Biolabs) according to the manufacturer’s protocol. The deglycosylation reaction was confirmed with two NuPAGE 4–12% Bis-Tris gels (Novex) in MOPS buffer. The first gel was transferred to a nitrocellulose membrane (Novex) with a Semi-Dry Blotter (Novex), blocked with carbo-free buffer (Vector Labs), labeled overnight at 4°C in 2 µg/mL biotinylated concavalin A (Vector), and probed with anti-biotin peroxidase (Vector) before imaging with Clarity Western Blot ECL Substrate (BioRad) in a FluorChemE unit (Cell Biosciences). The second gel was silver stained with Pierce Silver Stain Kit (Thermo Scientific) and imaged with the same unit. The remaining deglycosylated IgG and IgM was buffer-exchanged into PBS using 50 MWCO spin-x columns (Corning) and quantified based on A₂₈₀.

Anti-PEG IgG₁ (CH2076, Silver Lake Research) and anti-PEG IgM (AGP4, IBMS) were used as test Ab and anti-Biotin IgG₁ (Z021, Thermo Fisher) and anti-Vancomycin IgM (2F10, Santa Cruz) were used as control Ab. CH2076 was deglycosylated with rapid, non-reducing PNGase F (New England Biolabs) according to the manufacturer’s instructions. Deglycosylation was confirmed by SDS-PAGE gel with Coomassie stain and lectin-ELISA with biotinylated concanavalin A (B-1005, Vector Labs) and HRP-conjugated antibiotin IgG (033,720, Life Technologies). IgM was desialylated overnight with α2–3,6,8,9 Neuraminidase A (New England Biolabs) with 3 μL enzyme per 1 μg antibody at 37 °C. Desialylation was confirmed with lectin-ELISA with biotinylated elderberry bark lectin (B-1305, Vector Labs), biotinylated concanavalin A, and HRP-conjugated anti-biotin IgG. Due to the overnight incubation at 37 °C required to fully desialylate IgM and concerns it might denature IgM or otherwise reduce binding affinity to either PEG or the biogel matrix, we compared the trapping potency of desialylated IgM native IgM that was also incubated overnight at 37 °C.