Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach.
Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.
J K
JK
Russell
M P
MP
Hayes
J M
JM
Carter
B A
BA
Torres
B M
BM
Dunn
S W
SW
Russell
H M
HM
Johnson
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis / Q000502:physiology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D006224:Cricetinae; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology*; D051379:Mice; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D007371:Interferon-gamma