Horn Ivo R, Nielsen Marianne Jensby, Madsen Mette, Jacobsen Christian, Graversen Jonas Heilskov, Moestrup Søren K
Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
Eur J Haematol. 2003 Oct;71(4):289-93. doi: 10.1034/j.1600-0609.2003.00141.x.
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.
在血管内溶血过程中，血红蛋白（Hb）与触珠蛋白（Hp）结合，导致该复合物被巨噬细胞受体CD163内吞。在本研究中，我们采用噬菌体展示Fab抗体策略，以探究Hp与Hb之间的复合物形成是否会导致该复合物特异性抗原表位的暴露。通过对噬菌体-Fab文库进行Hp-Hb亲和筛选，我们分离出了一个针对该配体复合物的噬菌体克隆。对表达为重组蛋白的Fab部分进行表面等离子体共振分析，结果显示其与Hp-Hb具有高亲和力结合（KD = 3.9 nm），而未检测到与未复合的Hp或Hb有结合。该Fab抗体完全抑制了125I标记的Hp-Hb复合物与CD163的结合，并阻断了它们在CD163转染细胞中的摄取。总之，我们制备了一种特异性识别Hp-Hb复合物的受体阻断抗体。本研究除了为探究Hp与Hb结合时发生的变化提供了新的见解外，还为从血浆/血清中测量和去除Hp-Hb复合物提供了一种新的潜在工具。
