Exp Mol Med.  2002 Dec;34(6):411-418.

Characterization and epitope mapping of two monoclonal antibodies against human CD99

Affiliations
  • 1Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
  • 2Department of Pathology and Center for Molecular Medicine, Samsung Biomedical Research Insstitute, Sungkyunkwan University School of Medicine, Suwon, Korea. tjkim@yurim.skku.ac.kr
  • 3Department of Pathology, Ilsan Paik Hospital, Inje University College of Medicine, Koyang, Korea.
  • 4Department of Pathology, Hallym University College of Medicine, Chunchon, Korea.

Abstract

CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.

Keyword

antigens; CD; antibodies; monoclonal; antibody affinity; epitope mapping; epitopes; T lymphocyte

MeSH Terms

Amino Acid Sequence
Antibodies, Monoclonal/*immunology
Antigens, CD/*chemistry/*immunology
Blotting, Western
Cell Adhesion Molecules/*chemistry/*immunology
*Epitope Mapping
Epitopes/*chemistry/*immunology
Glutathione Transferase
Human
Molecular Sequence Data
Peptide Fragments/chemistry/immunology
Recombinant Fusion Proteins/chemistry/immunology
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