FIGURE 4.

Neutralizing antibodies are absorbed by different forms of the B2′B3. A–C, bar graphs represent the percentage of viable CHO cells after treatment for 24 h with 3.8 pm purified native TcdB_012. Cell viability was measured by a colorimetric reaction using WST-8 dye. To neutralize TcdB_012, the toxin was preincubated for 30 min with a 1:100 dilution of antiserum against B2′B3₀₁₂ (αB2′B3₀₁₂). The neutralization activities of the antiserum were absorbed by incubating the antiserum with B2′B3 for 30 min before the addition of purified native TcdB₀₁₂. A, this assay used 60 nm of B2′B3₀₁₂, B3₀₁₂, or B2′B3₀₂₇. The fragment of TcdB₀₁₂ from amino acid 1751 to 1853 was used at a concentration of 10 μm. B, 48 nm mutated B2′B3 (described in the legend to Fig. 1) was used in these experiments. C, this experiment was performed with 30 nm B2′B3₀₁₂ or 45 μm peptide spanning residues 1769–1787 of TcdB₀₁₂. Data are presented as mean (n = 3) ± S.D. (error bars). Asterisks indicate that antibody neutralization is significantly reduced. *, p < 0.05; **, p < 0.005.