Figure S1 - Trypanosoma cruzi trans-Sialidase in Complex with a Neutralizing Antibody: Structure/Function Studies towards the Rational Design of Inhibitors

Production of the mAb 13G9-TS immunocomplex. A) MonoQ-chromatogram of Protein A-purified hybridoma 13G9 supernatant. The mAb eluted as a single peak. B) TS reactivity of eluted and pass-trough proteins. Nitrocellulose membranes were spotted with TS-SAPA native (1) or heat-denatured (2). Upper panel was tested with flow through proteins, middle panel with the eluted peak and lower panel with an anti-SAPA mAb. Filters were developed with an HRP-labeled secondary antibody against mouse immunoglobulins. Note the absence of reactivity to the denatured protein by the 13G9 mAb (middle panel, spot 2) in contrast with the anti-SAPA mAb that recognizes a continuous epitope (lower panel). C) Purification of the Fab-TS complex through a Protein A affinity column. The retained protein corresponds to the Fc fraction. D) SDS-PAGE of the purified TS-Fab complex. E) Almost null remnant TS activity was found in the TS-Fab complex. (EPS)