Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies.
To characterize the nature of the immunoreactive transferrin receptor in human serum, antisera were developed to peptide sequences of the extracellular domain of human transferrin receptor between amino acids 107 and 120 and the intracellular domain between amino acids 40 and 54. Antisera against the extracellular domain exhibited reactivity against both purified intact receptor and immunopurified circulating receptor, whereas antisera against the intracellular domain reacted only with intact receptor. Using competitive binding enzyme-linked immunosorbent assays, transferrin receptor in ultracentrifuged sera from normal subjects and patients with sickle cell anemia could be detected with antisera against the extracellular but not the intracellular domain. When the pellet obtained by ultracentrifugation of these sera was assayed after solubilization in 1% teric (polyoxyethylene-9-lauryl ether), only 0.6% of total serum receptor was detected in normal subjects and 3.8% in subjects with sickle cell disease. Roughly equal amounts of this pelleted immunoactivity were detected with antibodies against the extracellular and intracellular domains. These results indicate that less than 1% of transferrin receptor in normal human sera is intact receptor consistent with an exosomal origin and that virtually all circulating transferrin receptor is in the form of a truncated extracellular domain.
Shih
Y J
YJ
Department of Medicine, Kansas University Medical Center, Kansas City 66160-7402.
Baynes
R D
RD
Hudson
B G
BG
Cook
J D
JD
D000595:Amino Acid Sequence; D000755:Anemia, Sickle Cell / Q000097:blood; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001667:Binding, Competitive; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010920:Placenta / Q000737:chemistry; D011990:Receptors, Transferrin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D000941:Antigens; D007106:Immune Sera; D010446:Peptide Fragments; D011990:Receptors, Transferrin