Protein g column

IgG fraction of the serum samples were isolated by Protein G column (GE Healthcare) according to the manufacturer’s instructions. For functional analyses, the in vivo formed FB–FB-autoantibody complexes were removed. To this end, FB-specific antibodies isolated by Protein G column from goat anti-FB antiserum were immobilized on NHS column (GE Healthcare). The patient’s and control’s IgG fractions were loaded onto the anti-FB column, the flow-through fraction was collected and tested for the presence of FB.

Human IgG was taken from Beriglobin (Behring). Murine IgG was obtained from pooled serum of healthy C57BL/6 mice (Charles River) of different age and sex by purification over a protein G column (GE Healthcare) according to the manufacturer’s instructions. For desialylation, 1 mg of human or murine IgG was incubated with 5U or 10,000U neuraminidase (NEB) for 24 h or 48 h, respectively, at 37 °C. For deglycosylation, human IgG was incubated with 500 U mg⁻¹ PNGase F (NEB) for 24 h at 37 °C. The efficiency of the enzymatic digestion was tested with a lectin blot. The digested IgG was purified over a protein G column (GE Healthcare) according to the manufacturer’s instructions and tested for endotoxin contamination using a LAL chromogenic endotoxin quantitation kit (Thermo scientific). Protein concentration was determined with the DC protein assay (Bio-Rad) and adjusted to 10 mg ml⁻¹. Immune complexes were obtained by heat aggregation of the IgG at 63 °C for 30 min.

The unmutated ancestor sequences (WEN-1^UA, WEN-3^UA) of the rCl13-neutralizing monoclonal antibodies WEN-1 and WEN-3 (Figure S1; [Eschli et al., 2007 (link), Seiler et al., 1998 (link)]) were identified by IgBlast (IgBlast) sequence analysis. All mutations deviating from the closest germline V(D)J heavy (HC) and light chain (LC) sequences were reverted. The corresponding cDNAs were synthesized (Genscript) and introduced into HC and LC expression cassettes for recombinant expression in a mouse IgG1 format (provided by Dr. Shozo Izui, University of Geneva). The antibodies were produced by transient co-transfection of the HC and LC expression plasmids in CHO cells (Protein Expression Core Facility, PECF, of the Swiss Federal Technical Highschool, EPFL, Lausanne, Switzerland). The antibodies were purified on an ÄKTAprime plus purification system using Protein G columns (GE healthcare). After 24 hours of PBS dialysis, the purified antibodies were quantified by IgG ELISA. MOPC-21 (BioXcell) served as isotype control antibody. The KL25-W104L, KL25-F106L and KL25-Y108S antibodies as well as a matching KL25 wild-type control antibody (Bruns et al., 1983 (link)) were obtained by analogous procedures, except that a mouse IgG2a expression format was used for these experiments.