Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody.
Anti-metatype antibodies have been described as antibodies which recognize ligand-induced conformational changes in the antibody variable region. Additionally, anti-metatype antibodies, produced by multiple immunizations with liganded high affinity monoclonal anti-fluorescein antibody 4-4-20, enhanced the lifetime of monoclonal antibody 4-4-20-fluorescein complex. To better understand the mechanism of the delayed dissociation rate, deuterium oxide was used to probe the liganded active site. The rate and extent of deuterium oxide-mediated fluorescence enhancement of bound ligand served to monitor the conformational dynamics of the active site in the presence and absence of anti-metatype antibodies. Results showed that anti-metatype antibodies reduced the rate and extent of deuterium oxide-mediated fluorescence enhancement of 4-4-20, a single-chain derivative of 4-4-20 (consisting of the variable domains and a polylinker), and idiotypically related monoclonal anti-fluorescein antibodies suggesting that anti-metatype stabilized the liganded active site. Size exclusion liquid chromatography was utilized to isolate the liganded antibody-anti-metatype complex. Liganded single chain antibody 4-4-20 was mixed with 10-fold molar excess anti-metatype Fab fragments, and a major complex eluted with an apparent M(r) 249,000. The apparent molecular weight of this complex inferred that one liganded single chain antibody was bound by five antimetatype Fab fragments. Spectral analysis confirmed these results and the characteristic delayed rate of ligand dissociation was also observed for the isolated complex. The results suggest that anti-metatype antibodies stabilize the liganded conformation by forming a large, stable, macromolecular complex.
Weidner
K M
KM
Department of Microbiology, University of Illinois, Urbana 61801.
Denzin
L K
LK
Voss
E W
EW
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002850:Chromatography, Gel; D003903:Deuterium; D019793:Fluorescein; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D008024:Ligands; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D014867:Water
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D008024:Ligands; D014867:Water; D003903:Deuterium; D019793:Fluorescein