This project is concerned with two aspects of the genetic and structural determinants of the affinity of immunoglobulins. The first is directed to the B cell response to synthetic peptides as epitopes in terms of the variety and selectivity of germline variable region genes used by anti-peptide antibodies. Hybridomas will be prepared which produce IgM and IgG antibodies. These will be purified and their intrinsic binding constants for a fluorescent conjugate of the homologous peptide will be measured by resonance energy transfer. This analysis will establish if affinity-restriction of IgM antibodies occurs in the case of peptide antigens. A comparison will also be made between linear and cyclic peptides with respect to immunogeneicity and affinity. Selected IgM-producing hybridomas will be subject to nucleotide sequencing to identify the germline VH and VL genes used for anti-peptide reactivity. The second aspect is directed to the use of site-directed mutagenesis to enhance the affinity of antibody. A cloned murine VH gene from one anti-dansyl hybridoma producing IgG of high affinity will be incorporated into a high-efficiency expression vector by recombinant technology. This plasmid construct will be used to transform E. coli under conditions that yield the VH product at a level of 10-20% of total bacterial protein. The VH product in the bacterial extract will be identified by Western blotting with a rabbit anti-serum to a FR2 sequence of the VH protein. After specific purification the VH product will be evaluated for binding affinity to dansyllysine either as a VH protein or after recombination with an appropriate L chain. Site- directed mutagenesis will be effected using a primer corresponding to the CDR3 region with nucleotide substitutions designed to provide selected amino-acid replacements.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI009492-30
Application #
3124567
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1975-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1992-03-31
Support Year
30
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104