Production of Novel Anti‐Recombinant Human Erythropoietin Monoclonal Antibodies and Development of a Sensitive Enzyme‐Linked Immunosorbent Assay for Detection of Bioactive Human Erythropoietin

Abstract

Erythropoietin (EPO) is a growth factor, regulating the proliferation and differentiation of erythroid progenitor cells. In this study, we generated five monoclonal antibodies (mAbs) that reacted specifically with recombinant human EPO (rhEPO). Epitope exclusion and other experiments showed that the mAbs obtained were divided into two groups, differing in recognition sites for rhEPO: group 1 mAbs recognize the N‐terminal region of rhEPO, whereas group 2 mAbs seem to recognize a conformation‐dependent epitope. Although most of the previously reported anti‐EPO antibodies that recognized the N‐terminal region of EPO lacked the EPO‐neutralizing activity, the group 1 mAbs obtained here had the rhEPO‐neutralizing activity. Therefore, the group 1 mAbs may be useful for future study on structure‐function relationship of EPO. One of the group 2 mAbs, 5D11A, showed the highest affinity for rhEPO with K_D value 0.52 nM and had the highest rhEPO‐neutralizing activity. Using this mAb, we developed a reproducible and sensitive enzyme‐linked immunosorbent assay for the quantification of bioactive rhEPO.

Keywords:

• Recombinant human erythropoietin
• Monoclonal antibodies
• Conformation‐dependent epitope
• Enzyme‐linked immunosorbent assay
• Validation
• Cell‐based potency assay

ACKNOWLEDGMENTS

We thank Yoko Tsuchiya and Yoshiaki Hagiwara (Immuno‐Biological Laboratories Co., Ltd.) for their skilled assistance in production of anti‐rhEPO mAbs. We are grateful to Mayumi Yumoto, Ryoko Takei and Yuko Kusakabe for their skilled technical assistance and Drs. Masayuki Kusano, Tomoyuki Tahara and Katsumi Tachibana for scientific discussions. We thank also Dr. Norio Komatsu (Department of Hematology, Yamanashi University) for the gift of UT‐7/EPO.