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Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis
1Department of Pathology, Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, MA 02118.
Journal of Immunology (Baltimore, Md. : 1950)
|June 15, 1990
Summary
Four anti-idiotypic monoclonal antibodies (mAB) recognize specific regions on mouse hybridoma protein 36-65. These antibodies map to complementarity-determining regions of the antibody
Area of Science:
- Immunology
- Molecular Biology
- Structural Biology
Background:
- Anti-idiotypic antibodies are crucial tools for understanding antibody structure-function relationships.
- Hybridoma protein 36-65, specific for p-azophenylarsonate, serves as a model antigen-binding protein.
- Hybridoma protein 36-71, a variant with 19 amino acid differences, lacks reactivity with the anti-idiotypic antibodies.
Purpose of the Study:
- To identify the specific regions of hybridoma protein 36-65 responsible for binding four anti-idiotypic antibodies (107, MB, AI, AD8).
- To investigate the contribution of both heavy (H) and light (L) chains to idiotope expression.
- To determine if the anti-idiotypic antibodies mimic the antigen structure.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to generate variants of the 36-65 H chain V region gene.
- Mutant proteins were expressed with either the 36-65 or 36-71 L chain in transfected hybridoma cells.
- Idiotypic characterization and hapten inhibition assays were performed on the mutant proteins.
Main Results:
- Reactivity of the anti-idiotypic antibodies primarily correlated with the H chain of hybridoma protein 36-65, with some contribution from the L chain.
- Idiotopes for antibodies 107, MB, and AI were mapped to the first and third complementarity-determining regions (CDRs) of the H chain V region.
- Idiotopes for antibody AD8 were mapped to all three CDRs of the H chain V region.
- Binding of all anti-idiotypic antibodies was hapten inhibitable.
- The effect of mutations on idiotope expression and antigen-binding affinity suggests the antibodies do not mimic the antigen.
Conclusions:
- The H chain, particularly the CDRs, plays a dominant role in the interaction with these anti-idiotypic antibodies.
- The L chain exhibits a modulatory role in idiotope recognition.
- None of the studied anti-idiotypic antibodies structurally mimic the p-azophenylarsonate antigen.