Analysis of functional domains of Vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies

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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.

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