Anti-idiotypic and anti-anti-idiotypic antibodies as highly specific tools in epitope and active site studies on human interferon-gamma.
Immunization of rabbits with F(ab')2 fragments of different monoclonal antibodies directed against human interferon-gamma yielded antisera with anti-idiotypic characteristics. Isolation of the anti-idiotypic fraction, resulting in a highly specific antiserum, allowed us to prove that out of six competing monoclonal antibodies directed against human interferon-gamma, only two really recognize the same epitope. The other monoclonal antibodies compete on the basis of steric hindrance, which is not surprising, because of the large difference in Mr between interferon-gamma and an immunoglobulin. The anti-idiotypes provided us also with a tool to study isolated epitopes on the human interferon-gamma molecule, a task which was previously not practicable. Exploration of the biological properties of these anti-idiotypes allows us to determine whether the investigated epitopes are involved in receptor binding. The production of an anti-anti-idiotypic antiserum not only proved that we generated real internal images, but also that these images conserved all of their properties, although with a decreased affinity in comparison with the original monoclonal antibody. As the former is a polyclonal antiserum, directed against a single epitope of the human interferon-gamma molecule, competition experiments yielded additional information on the relative position of three epitopes recognized by inhibiting monoclonal antibodies. These antisera will possibly open new ways for the affinity purification of interferon-gamma and perhaps for the treatment of autoimmune diseases.
Depla
E
E
Katholieke Universiteit Leuven, Laboratorium voor Biochemie, Belgium.
Louwagie
J
J
De Ley
M
M
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D001710:Biotin; D004686:Encephalomyocarditis virus / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*; D011817:Rabbits; D014407:Tumor Cells, Cultured
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D000939:Epitopes; D001710:Biotin; D007371:Interferon-gamma