title: Analysis of functional domains of Vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies.
abstract: Neutralizing monoclonal antibodies (mAbs) against Vibrio parahaemolyticus thermostable direct hemolysin (TDH) were used in probing the functional domains of this toxin. While pre-incubation of TDH with mAb 2A-13C inhibited further binding of TDH to erythrocytes, pre-incubation with another mAb 1-24 did not, indicating that mAb 1-24 epitope resides in a domain which is not involved in binding of TDH to erythrocytes. On the other hand after binding to erythrocytes, TDH could react with mAb 1-24 but poorly with mAb 2A-13C, indicating that the mAb 2A-13C epitope is masked, possibly by erythrocyte surface. As both antibodies are TDH-specific and do not react with TRH (TDH-related hemolysin), we used TDH/ TRH chimeric proteins to identify location of the epitopes for mAbs by inhibition ELISA as well as Western blotting. The results showed that the mAb 1-24 epitope resides on a region near the C-terminal of TDH (residues 99-139), while the mAb 2A-13C epitope resides on the N-terminal (residues 1-31). All these results suggested that, in TDH, the N-terminal region may be involved in binding process while the region near C-terminal may be involved in postbinding process.
authors.affiliation: Department of Bacterial Infections, Osaka University, Japan.
authors.forename: G
authors.initials: G
authors.lastname: Tang
authors.forename: T
authors.initials: T
authors.lastname: Iida
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: T
authors.initials: T
authors.lastname: Honda
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001665:Binding Sites; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship; D014736:Vibrio parahaemolyticus / Q000737:chemistry* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D000939:Epitopes; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C088609:thermostable direct hemolysin-related hemolysin protein, Vibrio parahaemolyticus; C068489:thermostable direct hemolysin