Characterization of the Antigenic Determinants of Cholera Toxin Subunits

Markel, Daiel E.; Hejtmancik, Kelly E.; Peterson, Johnny W.; Martin, Frank B.; Kurosky, Alexander

doi:10.1128/iai.25.2.615-626.1979

Cited by 10 publications

(2 citation statements)

References 23 publications

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“…Our initial data suggested a cross-reaction between LT-A and LT-B, since antisera to LT-A reacted with LT-B, but antisera to LT-B did not react with LT-A. These results were similar to previous reports on the antigenic cross-reactivity of the subunits of CT (10,25,36). However, antisera raised against more purified LT-A preparations reacted only with subunit A in Ouchterlony analysis and did not react with subunit B in the GM1 enzyme-linked immunosorbent assay (R. Ching and D. C. Robertson, unpublished observations).…”

Section: Resultssupporting

confidence: 91%

Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin

Gilligan¹,

Brown²,

Robertson³

1983

Infect Immun

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The antigenic relationships between heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (strain 286C2) and cholera toxin (CT) were examined by using antisera raised against LT and CT and specific antisera prepared against each subunit of both enterotoxins. Double immunodiffusion analysis revealed reactions of partial identity between the A subunits of LT and CT, as well as between the B subunits. Rabbit antisera raised against LT subunit A reacted with only subunit A, whereas rabbits immunized with LT subunit B produced antibodies which reacted with only subunit B. A high degree of CT neutralization was observed with antisera raised against LT. Data from neutralization assays with specific antisera to each enterotoxin showed that LT was more effectively neutralized by homologous anti-LT than CT (3.7-fold); however, anti-CT was only slightly more effective in neutralization of homologous CT compared with LT (1.9-fold). In contrast, antisera raised against the B subunit of CT (choleragenoid) exhibited significantly higher neutralization activity against CT than LT (5.8-fold); however, the amount of CT neutralized by anticholeragenoid was less (4.1-fold) than anti-CT. These results suggested that anti-CT serum contained neutralizing antibodies reactive with a shared determinant formed by interaction of the A and B subunits, whereas anti-LT and anti-choleragenoid sera did not. Sensitive solid-phase radioimmunoassays were developed to examine the affinity and degree of specificity involved in homologous and heterologous antigen-antibody interactions between LT, CT, their subunits, and specific antibodies. Only unlabeled LT competed with radiolabeled LT in polystyrene tubes coated with anti-LT, and only unlabeled CT competed with radiolabeled CT in tubes coated with anti-CT. However, when radiolabeled CT was incubated in tubes coated with anti-LT, competitive inhibition responses were observed with both unlabeled toxins. When radiolabeled LT was incubated with tubes coated with anti-CT, competitive inhibition responses were observed with both unlabeled toxins. Similar competitive inhibition responses were observed with the A subunits of LT and CT and with the B subunits using antisera specific for the subunits of each enterotoxin. Double immunodiffusion analysis and radioimmunoassay data supported the presence of unique and shared immunodeterminants in each subunit.

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Section: Resultssupporting

confidence: 91%

Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin

Gilligan¹,

Brown²,

Robertson³

1983

Infect Immun

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“…No monoclonal antibodies that reacted both with CT-A and CT-B were detected by Lindholm et al (24) or in our study. We believe that the evidence for shared epitopes on CT-B and CT-A is inconclusive and that the presence of antibodies to CT-B in hyperimmune sera prepared against purified CT-A (8,17,26) is more likely due to the presence of highly immunogenic CT-B as a trace contaminant in the antigen.…”

Section: Resultsmentioning

confidence: 97%

Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits

Holmes¹,

Twiddy²

1983

Infect Immun

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Seventeen selected hybridoma cell lines that produced monoclonal antibodies against cholera enterotoxin (CT) were isolated and characterized. All of the monoclonal antibodies contained the kappa light chain; 14 were of the immunoglobulin Gl (IgGl) isotype and 3 were IgG2a. The 17 monoclonal antibodies were divided into a minimum of seven different specificity groups based on their abilities to bind to the following purified test antigens in solid-phase radioimmunoassays: CT, the A and B polypeptides of CT (CT-A and CT-B, respectively), and the heat-labile enterotoxins designated LTh and LTp from Escherichia coli. The binding of these antibodies to the following subunits and fragments of CT was also determined in Western blots: pentameric CT-B, monomeric CT-B, intact CT-A, and the Al fragment of CT-A. Each of the monoclonal antibodies was tested for neutralization of CT and for precipitation with CT in immunodiffusion tests. Antigenic determinants were identified on CT that were not present either on CT-A or CT-B. One class was unique for CT and another was shared with LTh and LTp. Antibodies directed against these holotoxin-specific determinants had no neutralizing activity. Most of the monoclonal antibodies that reacted strongly with CT-A or CT-B also reacted strongly with CT holotoxin; however, one class of antibody reacted strongly with CT-A but weakly with CT. Among the monoclonal antibodies against CT-A or CT-B, some were specific for CT and others crossreacted with LTh and LTp or with LTh only. The most potent neutralizing antibodies were against CT-B, and all of our monoclonal antibodies against CT-B had some neutralizing activity. In contrast, only some of the monoclonal antibodies against CT-A had neutralizing activity, and their specific activities were low. We found no direct correlations between the ability of monoclonal antibodies to neutralize CT and to cross-react with LTh or LTp. None of the epitopes recognized by our monoclonal anti-CT antibodies was present on CT-A and CT-B.Cholera enterotoxin (CT) is a heat-labile enterotoxin produced by Vibrio cholerae (9). It has an essential role in the pathogenesis of cholera and causes secretory diarrhea by activating adenylate cyclase in the mucosal epithelium of the small intestine (13). CT is closely related to the heat-labile enterotoxin (LT) of Escherichia coli (34). The mode of action of LT is similar to that of CT, and LT has been implicated in the pathogenesis of secretory diarrhea in humans and in animals (13).Both CT and LT have been purified to homogeneity (3,9,18,22), and the structural genes for both toxins have been cloned (30,35,37). Each toxin is composed of two different polypeptide subunits designated A and B. Each holotoxin contains one A polypeptide and five copies of the B polypeptide held together by noncovalent bonds (14,15). Comparisons of primary structures demonstrated that the A and B polypeptides from CT have extensive homology with those from LT (6,36). Hyperimmune antisera prepared against either of the purified toxins will ne...

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Isolation and characterization of a precursor form of the ‘A’ subunit of cholera toxin

Duffy¹,

Peterson²,

Kurosky³

1981

FEBS Letters

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Characterization of the Antigenic Determinants of Cholera Toxin Subunits

Cited by 10 publications

References 23 publications

Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin

Immunological relationships between cholera toxin and Escherichia coli heat-labile enterotoxin

Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits

Isolation and characterization of a precursor form of the ‘A’ subunit of cholera toxin

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