Recombinant protein a

Protein A from Staphylococcus aureus (P3838), biotinylated native Protein A (P2165), and recombinant Protein A (P7837, expressed in E. coli), as well as human serum albumin (HSA, A9511), bovine serum albumin (BSA, A3059), human thrombin (89223 Fluka), and human immunoglobulin G (IgG, I4506) were purchased from Sigma-Aldrich (Germany). Human IgG-Fc fragment (009–0103) and human IgG-Fab fragment (009–0105) were from Rockland Immunochemicals, Inc. (Ireland). Recombinant Protein G and Protein L (Pierce 21193 and 21189, expressed in E. coli) were purchased from Fisher Scientific (Germany). Superparamagnetic Dynabeads M-270 Streptavidin (Strep-MB) were purchased from Invitrogen/Life Technologies (USA). According to the manufacturer’s instructions, these streptavidin-coated magnetic beads were used for immobilization of biotinylated native Protein A (P2165) as the target protein for aptamer selection to obtain Protein A-modified Strep-MB (Protein A/Strep-MB). PCR components like 10 × reaction buffer, 25 mM MgCl₂ and HOTFire polymerase were purchased from Solis BioDyne (Estonia). 100 mM stock solutions of dNTPs were from GE Healthcare (Germany).

SPR assays were performed using a Biacore^™ X100 instrument (GE Healthcare, Little Chalfont, UK). First, a protein A sensor chip was prepared by immobilizing recombinant protein A (Sigma) in both flow cells of a CM5 sensor chip to 5000 RU using the Amine Coupling Kit (GE Healthcare). VRC01 samples were diluted in HBS‐EP+ buffer and captured onto the Protein A surface (flow cell 2) to the levels around 240RU (for FcγRI analysis) or 330RU (for FcγRIIIa) or 680RU (for FcγRIIa and FcγRIIb). Recombinant human FcγR ectodomains (R&D) were injected over both flow cells at 25 °C for 135 s at 40 μL/min (FcγRI), 40 s at 50 μL/min (FcγRIIIa when tested against fucosylated antibodies), 90 s at 40 μL/min (FcγRIIIa when tested against afucosylated antibodies) or 30 s at 45 μL/min (FcγRIIa and b). The FcγR–VRC01 complexes were removed with two 90‐s injections of 10 mm glycine–HCl (pH 1.5). If the regeneration was not fully successful, the first injection was replaced with 10 mm glycine–HCl–3.5M MgCl₂ (pH 1.5) buffer. Receptors were analysed at the following concentrations: FcγRI—2–240 nm (480 nm for VRC01aglyco); FcγRIIIa—0.125–4 μm (12–800 nm for afucosylated antibodies VRC01_ΔXF and VRC01_Gal); and FcγRIIa and FcγRIIb—0.5–8 μm. Half‐life of antibody–receptor complexes was calculated from dissociation rate constants according to the formula: T_1/2 = ln2/k_off.