Figure 2.

Immunogenicity of recombinant SP antigens. (A–F) In all, 6- to 8-week-old female BALB/C mice (n = 5) were vaccinated with DTT-SP₄ or DTSP formulated in Alum and CpG three times at 10-day intervals. (A–C) Sera were collected 5 days after the final injection to assess antibody response. (A) Total IgG antibody reaction against SP was tested by ELISA. Sera were diluted 1:100. (B) Total IgG antibody titers were expressed as logarithm₁₀ reciprocals of the highest dilution giving twice the absorbance of the PBS sera. Sera were serially diluted from 1:100 to 1:4,096. (C) The level of serum IgG subclass of antibodies in response to SP at a 1:100 dilution. (D–F) Splenocytes were harvested 7 days or 45 days after the third immunization and stimulated with SP for 72 h in vitro. (D,E) Splenocytes were isolated 7 days after the final vaccination. (D) The absorbance of stimulated cells at 450 nm in the presence of 10 μL CCK-8 solution. (E) SIs were calculated using the ratio of the OD of stimulated cells to that of unstimulated cells at 450 nm. (F) Forty-five days after the last vaccination, SI of indicated splenocytes was determined. These data are presented as means ± SD. ^****p < 0.00001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05, ns, not significant, Student's t-test.