title: Molecular cloning of murine monoclonal anti-idiotypic Fab.
abstract: Anti-idiotypic antibodies (Ab2) binding to idiotopes on antibodies with various antigen binding specificities (Ab1) are potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer, and viral, bacterial, or parasitic infections. Furthermore, Ab2 are useful probes for the characterization of receptor/ligand interactions. Thus far, Ab2 production has been limited to the isolation of polyclonal Ab2 from immune sera or monoclonal Ab2 from hybridoma supernatants. However, both approaches have produced a limited number of Ab2. As an alternative approach, we demonstrate here the production of Ab2-Fab by using repertoire cloning. Using HIV-1 as a model system, the Ab2-Fab were generated from the spleens of mice immunized with the virus-neutralizing and syncytia-inhibiting anti-HIV-1 monoclonal antibody 0.5 beta. A bacteriophage lambda vector system was used to express a combinatorial library in Escherichia coli. Iodinated 0.5 beta was used to identify 17 Ab2-Fab clones. DNA sequence analysis of five clones revealed three similar kappa and Fd combinations. The Ab2-Fab bound with high affinity (3.5-6.5 x 10(9) liters/mol) specifically to the Ab1 and not to isotype-matched antibodies with unrelated specificities. The three Ab2-Fab probably bind to the same idiotope on the Ab1 as demonstrated in cross-competition binding studies. The Ab2-Fab inhibited binding of the Ab1 to antigen, and therefore, may functionally mimic the epitope defined by the Ab1. Repertoire cloning of Ab2-Fab may facilitate the generation of Ab2 that have potential as modulators of immune responses against various antigens.
authors.affiliation: Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104.
authors.forename: Y
authors.initials: Y
authors.lastname: Kasai
authors.forename: D
authors.initials: D
authors.lastname: Herlyn
authors.forename: M
authors.initials: M
authors.lastname: Sperlagh
authors.forename: H
authors.initials: H
authors.lastname: Maruyama
authors.forename: S
authors.initials: S
authors.lastname: Matsushita
authors.forename: A J
authors.initials: AJ
authors.lastname: Linnenbach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007140:Immunoglobulin Fab Fragments