Generation of a haptoglobin-hemoglobin complex-specific Fab antibody blocking the binding of the complex to CD163.
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.
Horn
Ivo R
IR
Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
Nielsen
Marianne Jensby
MJ
Madsen
Mette
M
Jacobsen
Christian
C
Graversen
Jonas Heilskov
JH
Moestrup
Søren K
SK
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
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