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.
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.
Tam
Frankie C H
FC
Clinical Immunology Unit, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong.
Ma
Chun Hung
CH
Leung
Danny T M
DT
Sutton
Brian
B
Lim
Pak-Leong
PL
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
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