FIGURE 5.

Two novel neutralizing epitopes are present in the RTX domain. A, two representative neutralizing scAbs were converted to chimeric IgG1/κ antibodies, in which the murine variable regions are appended by human constant domains. These were transiently expressed in CHO-K1 cells and purified by (NH₄)₂SO₄ precipitation and protein A affinity chromatography. SDS-PAGE (4–20% gradient gel, 2 μg loaded) shows high purity and expected size of the recombinant IgG as follows: 25 kDa (light chain) and 50 kDa (heavy chain) when reduced and 150 kDa under nonreducing conditions. ELISA demonstrates the ACT domain specificity of the M2B10 (B) and M1H5 (C) IgG antibodies. Microtiter plates were coated with ACT or ACT domains at equimolar concentrations, followed by serial dilution of antibody from 1 nm, and followed by detection with anti-human Fc antibody-HRP conjugate. D, competition ELISA determined that the M2B10 and M1H5 antibodies bind novel nonoverlapping epitopes. A 200-fold molar excess (20 nm) of previously described murine mAbs (3D1, 2A12, 10A1, 2B12, 9D4, 6E1, 7C7, and 1H6) (47) or scAb versions of M1H5 and M2B10 were mixed with M2B10 and M1H5 IgG (0.1 nm) and incubated on ACT-coated ELISA plate, with bound M2B10 or M1H5 detected as above. The absorbance was normalized to that of M2B10 or M1H5 with no competitor; absorbance significantly <1.0 indicates competition between the antibody pair.