Characterization and epitope mapping of two monoclonal antibodies against human CD99.
CD99 plays a 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.
Gil
Min Chan
MC
Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea.
Lee
Mi-Hong
MH
Seo
Jeong-In
JI
Choi
Yoon-La
YL
Kim
Min-Kyung
MK
Jung
Kyeong Cheon
KC
Park
Seong Hoe
SH
Kim
Tae Jin
TJ
D000071443:12E7 Antigen; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000737:chemistry* / Q000276:immunology*; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D005982:Glutathione Transferase; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology
D000071443:12E7 Antigen; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase