Analysis of functional domains of Vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies.
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.
Tang
G
G
Department of Bacterial Infections, Osaka University, Japan.
Iida
T
T
Yamamoto
K
K
Honda
T
T
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
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