Immunochemical analysis of monoclonal antibodies to human erythropoietin.
We recently reported the development of three monoclonal antibodies (MoAbs) to biologically active human erythropoietin (Ep). In the present study, we investigated the epitope specificity of these three antibodies, as well as their reactivity with Eps derived from species other than man. All three antibodies reacted with the Ep polypeptide itself, rather than with its carbohydrate moieties. Moreover, all three antibodies recognized separate nonoverlapping epitopes. Further studies with reduced/alkylated Ep and with sodium dodecyl sulfate-denatured Ep suggested that two of the MoAbs, anti-Ep-2 and anti-Ep-16, were specific for conformational, nonlinear determinants on the Ep molecule, whereas the third MoAb, anti-Ep-26, appeared to recognize a linear epitope. However, anti-Ep-26 did not react with synthetic peptides representing the 26 amino-, the 99-129 mid-region, or the 10 carboxy-terminal residues of Ep, nor with trypsin-, chymotrypsin-, or V8 protease-digested fragments of Ep. When tested with Ep from different species, the neutralizing capabilities of the three MoAbs were clearly different. Comparing their effectiveness against baboon, ovine and murine Ep, antibody 2 was most effective at neutralizing baboon Ep, antibody 16 was most effective against murine Ep, and antibody 26 showed little reactivity with any of these nonhuman Eps. Because these various Eps readily stimulate across species barriers, it is likely that the receptor binding domain on Ep has remained relatively conserved during evolution. Our results therefore suggest that the neutralizing capacity of our three anti-Ep MoAbs is caused not by binding directly to the Ep receptor binding domain on Ep, but by binding to distant regions, causing conformational changes in Ep, or by binding to regions close to the binding site, steric hindrance.
Wognum
A W
AW
Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, Canada.
Lansdorp
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PM
Krystal
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G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002918:Chymotrypsin; D000939:Epitopes / Q000276:immunology*; D004921:Erythropoietin / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D010215:Papio / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases; D012756:Sheep / Q000276:immunology; D013045:Species Specificity; D014357:Trypsin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D004921:Erythropoietin; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin