FIGURE 7.

RTX domain is immunodominant and elicits neutralizing antibodies. A, immunization with ACT yields sera preferentially recognizing the RTX domain. Purified domains were coated at equal moles on microtiter plates, with sera serially diluted starting at 1:200. The average EC₅₀ for individual domains is shown. Error bars are the standard deviations of the EC₅₀ value among four mice. B, immunogenicity of purified domains. Mice were immunized with intact ACT or individual domains, with the serum EC₅₀ for ACT measured by ELISA after the first boost (6 weeks) or second boost (8 weeks). C, sera from mice immunized with the ACT and RTX₉₈₅ domain neutralize cAMP intoxication similarly. Sera from each immunization group at a 1:400 dilution were incubated with 125 ng/ml ACT in DMEM before adding to J774A.1 cells. Intracellular cAMP levels were measured by cAMP ELISA, divided by the total protein concentrations, and normalized to cells treated with ACT alone, as in Fig. 4. pre indicates baseline sera collected prior to immunization. Statistical significance was determined by one-way analysis of variance with Tukey's test; **, p ≤ 0.01; ***, p ≤ 0.001. For all panels, * indicates an acylated domain. n.s. indicates not significant (p > 0.05).