Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.
Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.
Bedzyk
W D
WD
Department of Microbiology, University of Illinois, Urbana 61801.
Weidner
K M
KM
Denzin
L K
LK
Johnson
L S
LS
Hardman
K D
KD
Pantoliano
M W
MW
Asel
E D
ED
Voss
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D019793:Fluorescein; D005452:Fluoresceins / Q000378:metabolism; D007130:Immunoglobulin Idiotypes; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014867:Water
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007130:Immunoglobulin Idiotypes; D008024:Ligands; D011994:Recombinant Proteins; D014867:Water; D019793:Fluorescein