title: Use of a human recombinant immunoglobulin G1 CH3 domain as a probe for detecting alternatively folded human IgG in intravenous Ig products.
abstract: It has been previously reported that intravenous immunoglobulin (IVIg) contains alternatively folded (aggregation-prone) monomeric immunoglobulin (Ig) G molecules. These alternatively folded IgG molecules may act as precursors for Fc-Fc-mediated dimerization and/or aggregation in IVIg. To study this phenomenon, we set up a fluid-phase binding assay using an acid-shocked (pH 2.5) recombinant human IgG1 CH3 domain as a probe in combination with size-exclusion chromatography. Three IVIg products and a recombinant IgG1 antibody were analyzed. Besides CH3 probe binding to monomeric IgG derived from all IVIg products, the CH3 probe also bound to IgG4 half-molecules. This IgG4 binding could be distinguished from binding to IgG molecules on the basis of molecular weight. In contrast, no CH3 probe binding to IgG from the recombinant IgG1 antibody was observed. After acid-induced aggregation of either IVIg or a recombinant IgG1 antibody, CH3 probe binding to oligomeric complexes was observed, but no longer to monomeric IgG, demonstrating that the alternatively folded monomeric IgG molecules had oligomerized. Our results indicate that the tested IVIg products contain traces of alternatively folded IgG molecules within the "normal" monomeric IgG fraction. Furthermore, we conclude that the fluid-phase binding assay is sensitive to detect these alternatively folded IgG molecules in IVIg.
authors.affiliation: Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Theresa
authors.initials: T
authors.lastname: Guhr
authors.forename: Ninotska
authors.initials: N
authors.lastname: Derksen
authors.forename: Rob
authors.initials: R
authors.lastname: Aalberse
authors.forename: Theo
authors.initials: T
authors.lastname: Rispens
mesh_terms: D015153:Blotting, Western; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry; D016756:Immunoglobulins, Intravenous / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D016756:Immunoglobulins, Intravenous; D011994:Recombinant Proteins