Endobulin

As a source of human polyclonal IgG obtained from healthy individuals, we used pooled immunoglobulin G preparation for therapeutic intravenous use (IVIG, Endobulin, Baxter International, Deerfield, IL, USA). We also used a previously described human recombinant monoclonal IgG1 (Ab21) [30 (link)].
A standard solution of hyperoxidized heme (heme-ox) was prepared by treatment of 1 mM of heme (Fe³⁺) or hemin (ferriproptoporphyrin IX, Frontier Scientific Inc., Logan, UT, USA) dissolved in 0.05 N NaOH with 25 mM final concentration of H₂O₂ (Merck, Darmstadt, Germany) in 0.05 N NaOH. Formation of gas bubbles after addition of H₂O₂ signified the catalytic degradation of peroxide to H₂O and O₂. For generation of oxidized species of heme, the samples were incubated in the presence of hydrogen peroxide for at least 10 min before any further use. In another setting, 1 mM hemin solution in NaOH was exposed to increasing concentrations (0.015–1000 mM, dilution step of 3) of hydrogen peroxide or sodium hypochlorite (Sigma-Aldrich, St. Louis, MO, USA). As a control, 1 mM solution of Zn(II) protoporphyrin IX (Frontier Scientific Inc.) in 0.05N NaOH was treated with 25 mM H₂O₂. In another case, heme (Fe³⁺) and heme-ox at 1 mM were reduced by incubation in the absence or in the presence of 10 mM ascorbic acid (Sigma-Aldrich) for 30 min at RT and then used for treatment of polyclonal IgG.

Human and bovine oxidized Hb (or methemoglobin, metHb) equine myoglobin and equine cytochrome c were all obtained in lyophilized form (Sigma-Aldrich, St. Louis, MO, USA). Fresh stock solutions of hemoproteins in PBS were prepared immediately before use and kept at ice in dark. Apo-Hb was prepared from human Hb by using a procedure of heme extraction by acidified acetone as described in^{93 (link)}. Therapeutic intravenous immunoglobulin (IVIg, Endobulin, Baxter, USA) that consists of pooled human IgG purified from >3000 blood donors, was used as a source of polyclonal IgG from healthy individuals. A monoclonal IgG1 antibody—Ab21 that was previously identified as able to acquire polyreactivity upon interaction with heme—was used as a representative heme-sensitive antibody throughout the study^{59 (link)}. Sera obtained from anonymous healthy blood donors (under the ethical convention between INSERM with Etablissement Français du Sang—15/EFS/012) were used as a source of normal human IgG.

Streptococcal cell wall extracts were isolated as previously described26 (link). Briefly, overnight S. pyogenes cultures were diluted 1:10 in fresh THB and grown to an A₆₀₀ of 0.4–0.8. The cells were pelleted and resuspended in 1/50th volume of 10 mM Tris-HCl (pH 8.0) containing 30% raffinose, 100 U/ml mutanolysin, 1 mg/ml lysozyme and 10% Protease Inhibitor Cocktail Set III (Calbiochem), and incubated for 3 h at 37 °C with occasional inversion. The cells were pelleted and the supernatant (cell wall extract) was dialysed into PBS overnight and concentrated by centrifugation (10,000 MWCO). For immunoblotting, proteins were separated on 7% tris-acetate (TA) gels (Invitrogen) and transferred to Hybond LFP membranes (GE Healthcare). Membranes were blocked with 5% skimmed milk prior to the addition of 4 μg/ml IVIG (Endobulin^®, Baxter). Bound antibodies were detected using a 1:80,000 dilution of HRP-conjugated goat anti human IgG (Sigma-Aldrich) and the ECL prime detection system (GE Healthcare).