Altered hapten recognition by two anti-digoxin hybridoma variants due to variable region point mutations.
Two spontaneous variants of the murine anti-digoxin antibody-producing hybridoma cell line 26-10 were isolated by two-color fluorescence-activated cell sorting on the basis of altered hapten binding. The variable region sequences of the antibodies produced by the mutant lines revealed that each contains a single amino acid change in the heavy chain second complementarity determining region. A Tyr to His change at position 50 leads to a 40-fold reduction in affinity for digoxin. A Ser to Phe mutation at position 52 results in a 300-fold reduction in affinity for digoxin. A competition assay involving 33 digoxin analogues was used to examine the specificity of hapten binding of 26-10 and the two mutant antibodies. The position 50 mutant has a distinct specificity change; it exhibits a preference for digoxin congeners containing a hydroxyl group at the steroid 12 position, whereas the 26-10 parent does not. The affinities of all three antibodies for hapten are progressively lowered by substitutions of increasing size at the digoxin steroid D ring 16 position. Although 26-10 binds digoxin and its genin form equally, 12 and 16 steroid position substitutions which lower affinity also confer a preference for a sugar at the steroid 3 position. These results suggest that position 50 contributes to specificity of the antibody and that alterations of the hapten can lead to differences in recognition, possibly through a shift in hapten orientation within the binding site.
Schildbach
J F
JF
Department of Medicine, Massachusetts General Hospital, Boston.
Panka
D J
DJ
Parks
D R
DR
Jager
G C
GC
Novotny
J
J
Herzenberg
L A
LA
Mudgett-Hunter
M
M
Bruccoleri
R E
RE
Haber
E
E
Margolies
M N
MN
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D004076:Digoxigenin / Q000276:immunology; D004077:Digoxin / Q000031:analogs & derivatives / Q000276:immunology*; D005803:Genes, Immunoglobulin; D006241:Haptens; D006825:Hybridomas; D007130:Immunoglobulin Idiotypes / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010042:Ouabain / Q000276:immunology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D006241:Haptens; D007130:Immunoglobulin Idiotypes; D010042:Ouabain; D004077:Digoxin; D004076:Digoxigenin; C005003:gitoxin