title: Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163.
abstract: During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage-display Fab antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic epitopes specific for the complex. By Hp-Hb-affinity screening of a phage-Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp-Hb, whereas no binding was measured for non-complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I-labeled Hp-Hb complexes to CD163 and blocked their uptake in CD163-transfected cells. In conclusion, we have raised a receptor-blocking antibody specifically recognizing the Hp-Hb complex. In addition to provide new insight into the changes occurring when Hp and Hb bind, the present study provides a new potential tool for measuring and removal of Hp-Hb complexes from plasma/serum.
authors.affiliation: Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Ivo R
authors.initials: IR
authors.lastname: Horn
authors.forename: Marianne Jensby
authors.initials: MJ
authors.lastname: Nielsen
authors.forename: Mette
authors.initials: M
authors.lastname: Madsen
authors.forename: Christian
authors.initials: C
authors.lastname: Jacobsen
authors.forename: Jonas Heilskov
authors.initials: JH
authors.lastname: Graversen
authors.forename: Søren K
authors.initials: SK
authors.lastname: Moestrup
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D000939:Epitopes; D006242:Haptoglobins / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D008024:Ligands; D019151:Peptide Library; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D000939:Epitopes; D006242:Haptoglobins; D006454:Hemoglobins; D007128:Immunoglobulin Fragments; D008024:Ligands; D019151:Peptide Library; D011956:Receptors, Cell Surface