Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure.

We have isolated and mapped the rHuEPO epitopes for three noncompeting anti-EPO monoclonal antibodies (MoAbs). The MoAb 9G8A recognizes a linear epitope that includes amino acids 13, 16, and 17. MoAb F12 recognizes a conformational epitope that includes amino acids 31 through 33, 86 through 91, and 138. MoAb D11 recognizes a conformational epitope that includes amino acids 64 through 78 and 99 through 110. MoAb D11 neutralizes rHuEPO activity which suggests that its epitope may contain the receptor binding domain. Analysis of the effect of mutations on folding allowed the identification of buried residues, alpha-helical, and non alpha-helical regions. This data along with epitope mapping data of anti rHuEPO monoclonals was used to model rHuEPO protein structure. A model consistent with the data is a 4-helix bundle with short and long interconnecting loops.