Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDSpolyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.

A murine monoclonal antibody (MAb), VTm1.1, specifically recognizing and neutralizing Shiga toxin 2 (Stx2), was obtained. To prevent a humoral response against murine antibody when used clinically, a humanized antibody was constructed by combining the complementarity-determining regions of VTm1.1 with human framework and constant regions. In addition, several amino acids in the framework were changed to improve the binding affinity of the antibody and further reduce its potential immunogenicity. The humanized antibody, TMA-15, recognized the B-subunit of Stx2 and had affinity for Stx2 of 3.3 x 10(-9) M, within two-fold of that of the original murine antibody. TMA-15 neutralized the cytotoxicity of Stx2 and several different Stx2 variants in vitro, and it completely protected mice from death in a Stx2-challenged mice model. These results suggest that TMA-15 will have clinical potency in Stx-producing Escherichia coli infections, including E. coli O157 infections.

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