ABSTRACT
By repeatedly introducing hydrophilic polyethylene glycol (PEG) spacer (2) onto affinity resin bearing a bioactive peptide (1/2 secretory leukocyte protease inhibitor, 1/2SLPI) as a ligand, the adsorption of nonspecific binding proteins was effectively reduced and the purification efficacy of elastase, which is one of the target molecules for 1/2SLPI, from a protein mixture was improved. Moreover, using this resin, we also successfully detected L-plastin, as an endogenous target molecule for SLPI, from HL-60 cell lysate.
Subject(s)
Peptide Fragments/chemistry , Proteins/chemistry , Adsorption , Chromatography, Affinity/methods , HL-60 Cells , Humans , Ligands , Membrane Glycoproteins , Microfilament Proteins , Phosphoproteins/chemistry , Phosphoproteins/isolation & purification , Polyethylene Glycols/chemistry , Proteinase Inhibitory Proteins, Secretory , Secretory Leukocyte Peptidase Inhibitor , Serine Proteinase Inhibitors/chemistry , Structure-Activity Relationship , Subcellular Fractions/chemistry , Surface PropertiesABSTRACT
Shiga toxin 2 (Stx2) is a major pathogenic factor in Shiga toxin-producing Escherichia coli (STEC) infections. Some factor that neutralizes Stx2 in vitro had been shown to be specifically present in human serum and we recently identified it as human serum amyloid P component (HuSAP). Here, we report the role of HuSAP in STEC infections. HuSAP could not rescue Stx2-challenged mice from death, and it instead reduced the efficacy of the Stx2-neutralizing humanized monoclonal antibody TMA-15 when a lower dose of TMA-15 was injected to the mice. By contrast, the efficacy of TMA-15 at a higher dose was uninfluenced by the presence of HuSAP. These findings suggest that HuSAP acts as a carrier protein of Stx2 rather than as a Stx2-neutralizing factor in the human circulation and that passive immune therapy with Stx2-neutralizing antibodies such as TMA-15 is useful to prevent severe complications associated with STEC infections even in the presence of HuSAP.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Carrier Proteins/physiology , Escherichia coli Infections/etiology , Escherichia coli/pathogenicity , Serum Amyloid P-Component/physiology , Shiga Toxin 2/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Binding, Competitive , Escherichia coli Infections/microbiology , Escherichia coli Infections/therapy , Female , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Shiga Toxin 2/immunology , Shiga Toxin 2/metabolism , Shiga Toxin 2/toxicityABSTRACT
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.