Induction of polyclonal and monoclonal antibody responses to cholera toxin by the synthetic peptide approach

Abstract

The induction of an antibody response to cholera toxin (CT) was studied by using the synthetic peptide approach. Two peptides, corresponding to the amino acid sequences from residues 57 to 69 (CTBP 1) and 47 to 60 (CTBP 2) of the cholera toxin B subunit, were synthesized by the solid-phase method. These peptides were primarily chosen on the basis of their hydrophilicity and sequence identity with the B subunit of E. coli toxin (LT h). Synthesized peptides were coupled to carrier proteins through additional cysteine residues at the carboxyl (CTBP 1) or amino terminal ends (CTBP 2). Rabbit antisera to the peptide-carrier conjugates were found to react with the free peptides as well as intact CT, its B subunit and LT h as determined by the conventional enzyme-linked immunosorbent assay (ELISA). On the other hand, anti-peptide sera failed to react with CT and LT h in GM 1 (ganglioside)—ELISA, thereby suggesting the possible involvement of ctbp 1 and CTBP 2 peptide regions of the toxin molecule in GM 1 receptor binding. Both anti-peptide sera possessed rather weak toxin neutralizing activity in the rabbit ileal loop assay. However, such activity was statistically significant (0.02 < P < 0.05) only in the case of anti-CTBP 2 serum. Similar results were also obtained with mouse polyclonal anti-peptide sera. Ten mouse monoclonal antibodies were obtained against the CTBP 1 peptide, five of which reacted to CT, the B subunit and LT h in ELISA. Interestingly, one monoclonal showed strong reactivity against CT and LT h although it reacted very weakly against the immunizing peptide CTBP 1. It appears that the immunizing peptide probably exists in multiple conformers in the conjugated form, some of which may mimic more closely its structural features in the intact protein than in the free state. Results obtained in this study suggest that synthetic peptides can serve as useful probes for the structural analysis of CT or related toxins and may be useful in vaccine development.