Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells.
The terminal alpha anomeric Ga1NAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A,M., Jones N., Transfusion 31: 142-149, 1991] we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of these two MoAbs were hampered by the lack of availability of Gal/GalNAc related glycotopes. In order to use these antibodies as powerful probes to elucidate structural changes during life processes, we have characterized in detail the combining sites of these two MoAbs using enzyme-linked immunosorbent (ELISA) and inhibition assays with an extended glycan/ligand collection. From the results, it has been established that BRIC66 demonstrated multiple specificities and its reactivity towards glycotopes was defined as: Ga1NAc alpha1-->Ser/Thr (Tn) > or = Ga1NAc alpha1-->3(LFuc alpha1-->2)Gal (Ah) > Ga1NAcalpha1-->3Galbeta1-->4Glc (AL) > Ga1NAalpha1-->3Gal (A) GalNAc alpha1-->3GalNAc >> Gal or Glc. Another MoAb, BRIC111, mainly bound Tn-glycophorin. The best ligand for this MoAb was Tn-containing glycopeptides (M.W. < 3.0 x 10(3) Da) from asialo ovine salivary mucin (OSM), which was approximately 70 and 58 times more active than Ga1NAc and monomeric Ga1NAc alpha1-->Ser/Thr (Tn), respectively, suggesting that the active glycotopes present in glycophorin for BRIC111 binding also exist in OSM. The N-acetyl group at carbon-2 and configuration at carbon-2 and carbon-4 of the alpha anomeric Ga1NAc are required for the binding of either MoAb. Identification of these binding properties should aid in the selection of these MoAbs and the conditions required for biological studies and clinical applications.
Wua
Albert M
AM
Glyco-Immunochemistry Research Laboratory, Institute of Molecular and Cellular Biology, Taiwan. amwu@mail.cgu.edu.tw
Wub
June H
JH
Kuoa
Hsiang-Wei
HW
Herpa
Anthony
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism*; D001665:Binding Sites; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D006023:Glycoproteins; D009844:Oligosaccharides; C040286:Tn antigen