Heme-binding Protein Interactions and Antibody Polyreactivity

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.