Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis.

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The antigenic regions of Escherichia coli alpha-hemolysin were determined by antibody binding to cyanogen bromide (CnBr) fragments of this protein under denatured conditions. Alpha-hemolysin was isolated from filtered culture supernatants of a recombinant strain by a combination of trichloroacetic acid precipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Alpha-hemolysin was used to (i) produce polyclonal rabbit antisera and murine monoclonal immunoglobulin G (IgG) antibodies and (ii) generate CnBr fragments. Rabbit IgG and 13 murine IgG monoclonal antibodies (MAbs) were elicited to alpha-hemolysin as determined by enzyme-linked immunosorbent and immunoprecipitation assays. Antibodies bound to three specific CnBr fragments of alpha-hemolysin in Western blots (immuno-blots) from sodium dodecyl sulfate-polyacrylamide gels: CnBrII (encompassing residues [R] 2 to 160), CnBrV (R 425 to 892), and CnBrVI (R 893 to 1023). Five MAbs bound to CnBrII, seven MAbs bound to CnBrV, and one MAb bound to CnBrVI. These specific CnBr fragments are predicted to be hydrophilic and charged. There was no antibody binding to the highly hydrophobic CnBrIII (R 161 to 416). Similar binding patterns were observed when rabbit polyclonal anti-alpha-hemolysin IgG was used. Polyclonal antibodies to alpha-hemolysin readily inhibited hemolysis and its neutralization capacity was 4- to 64-fold more potent than neutralizing MAbs. The five MAbs that bind to CnBrII possessed hemolytic neutralizing activity to various degrees. In contrast, only three of seven MAbs that bind to CnBrV fragment exhibited neutralization capacity to various degrees; the MAb to CnBrVI did not exhibit this capacity. Based on these data, we predict that denatured alpha-hemolysin and its CnBrII and CnBrV fragments might be worthwhile immunoprophylactic candidates for the prevention of hemolysin-mediated E. coli tissue injury.

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