title: Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues.
abstract: Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen. Here, we investigated whether the problem was linker-related by changing the linker composition or by deleting it, but these attempts proved futile. Instead, we have constructed a recombinant Fab fragment of the antibody in bacteria that was carrier-specific. This suggests that constant regions are required for the carrier specificity, which presumably helps to mould the fine structure of the antibody combining site or in stabilizing such a structure. Consistent with this global effect is the finding that replacing specific residues in VH with germ-line residues, namely, VH49 glycine and VH30 threonine, both thought previously to be important for the carrier specificity, had no effect on the carrier specificity of the recombinant Fab.
authors.affiliation: Clinical Immunology Unit, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.
authors.forename: Frankie C H
authors.initials: FC
authors.lastname: Tam
authors.forename: Chun Hung
authors.initials: CH
authors.lastname: Ma
authors.forename: Danny T M
authors.initials: DT
authors.lastname: Leung
authors.forename: Brian
authors.initials: B
authors.lastname: Sutton
authors.forename: Pak-Leong
authors.initials: PL
authors.lastname: Lim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000918:Antibody Specificity; D000947:Antigens, Helminth / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D005998:Glycine / Q000737:chemistry; D006241:Haptens / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010767:Phosphorylcholine / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D017160:Trichinella spiralis / Q000276:immunology* / Q000378:metabolism
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D010767:Phosphorylcholine; D013912:Threonine; D005998:Glycine