Polypeptide fragments of horse cytochrome c activate a small subset of secondary B lymphocytes primed against the native protein.
Horse cytochrome c (cyt c) and two large, overlapping cyanogen bromide-cleaved fragments (1-80 and 66-104), together encompassing the entire length of the polypeptide chain, were examined for their abilities to stimulate into antibody production individual secondary B lymphocytes primed against the intact protein. T cell help was provided against the carrier protein, hemocyanin, to which cyt c and its peptides were conjugated by using glutaraldehyde. All the B cells activated by both of the fragments elicited antibodies that reacted with intact cyt c in enzyme-linked immunosorbent assay, whereas only a fraction of the antibodies elicited by the intact protein reacted with the peptides. However, in general, antibodies reactive with the polypeptide fragments, whether elicited by the intact protein or by the fragments, could not be effectively inhibited from binding plate-bound cyt c in enzyme-linked immunosorbent assay in the presence of soluble native cyt c. This indicates that these antibodies are specific for denatured forms of cyt c that apparently arise during the chemical coupling of cyt c to carrier molecules for immunization and/or during emulsification of the immunogen in adjuvant. Whereas, at most, 5% of the secondary B cells specific for native cyt c could be activated by the 1-80 fragment, even fewer were activated by the 66-104 fragment. Therefore, it is unlikely that smaller peptides which fail to assume native conformation would be effective. Antibodies elicited in vivo in a primary response to the 1-80 fragment also failed to bind native cyt c. These results suggest that linear peptides intended to mimic epitopes on globular proteins, and which have not been engineered to adopt native conformation, will not be very effective either as primary or as secondary vaccines for B cell activation.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D001402:B-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000276:immunology*; D005260:Female; D007143:Immunoglobulin Heavy Chains / Q000032:analysis; D007156:Immunologic Memory; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology*; D013154:Spleen / Q000166:cytology
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group; D007143:Immunoglobulin Heavy Chains; D010446:Peptide Fragments