The long term goal of this project is to determine the relationship between diversity and complementarity and to determine the repertoire and fine specificity of combining sites to a single well defined antigenic determinant, the site filling epitope of alpha(1 ->6)dextran. We have studied a series of hybridomas specific for alpha(1->6)dextran, characterized their antibodies immunochemically as to fine structure and idiotypic specificity, and determined the primary structures of their VH and VL. Modeling of cavity- and groove-type sites, will be continued to determine variations in the sites in relation to sequence differences. An ultimate objective is to determine the three dimensional xray structure of their combining sites. Studies with hybridomas have shown that they do not comprise the totality of antibody specificities seen in serum. Thus there is a hidden portion of the repertoire which can only be discovered by the use of multiple strains, varied conditions of immunization etc. This is important in determining the extent of germ line usage in evaluating the repertoires. Different VH and VL can generate similar binding specificities and the presence of N-linked carbohydrate in VH influences the strength of the antigen-antibody interaction; thus not only the amino acid sequence of the variable region but also the carbohydrate present there is important for determining binding. Site directed mutagenesis will be coupled with gene transfection techniques to evaluate the contributions of residues in VH and VL to specificity and binding. We have studied a monoclonal anti-blood group A in humans and mice and find that identity in fine structure can occur with different residues in the CDRs of the two species; this will be expanded to define the repertoire in two different species to anti-B as well anti-A in relation to identities and differences in CDRs and fine specificity of the sites. We shall compare the hybridoma repertoire in great detail with that produced in E. coli by the Lerner procedure with respect to cavity- vs groove-type sites, binding constants, site sizes, fine structure using various oligo- and fluorodeoxy oligosaccharides, and idiotype specificities. The findings will establish whether the two repertoires are drawn from the same universe of antibody combining sites.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019042-08
Application #
3128472
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1983-09-30
Project End
1996-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
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Wu, J H; Song, S C; Chen, Y Y et al. (1998) Multi-antennary Gal beta1-->4GlcNAc and Gal beta1-->3GalNAc clusters as important ligands for a lectin isolated from the sponge Geodia cydonium. FEBS Lett 427:134-8
Wu, A M; Song, S C; Chang, S C et al. (1997) Further characterization of the binding properties of a GalNAc specific lectin from Codium fragile subspecies tomentosoides. Glycobiology 7:1061-6
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Nickerson, K G; Tao, M H; Chen, H T et al. (1995) Human and mouse monoclonal antibodies to blood group A substance, which are nearly identical immunochemically, use radically different primary sequences. J Biol Chem 270:12457-65
Wu, A M; Song, S C; Wu, J H et al. (1995) Affinity of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin B4 for Gal alpha 1-->4 Gal ligand. Biochem Biophys Res Commun 216:814-20
Liao, J; Nickerson, K G; Bystricky, S et al. (1995) Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi. Infect Immun 63:4429-32
Chen, J; Borden, P; Liao, J et al. (1992) Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(1----6)dextrans with groove- or cavity-type combining sites. Mol Immunol 29:1121-9
Schiffer, M; Kabat, E A; Wu, T T (1992) Subgroups of Tcr alpha chains and correlation with T-cell function. Immunogenetics 35:224-34
Padlan, E A; Kabat, E A (1991) Modeling of antibody combining sites. Methods Enzymol 203:3-21

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