Radioimmunoassay for the Antigenic Determinants of Cholera Toxin and Its Components

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

doi:10.1128/iai.17.3.621-628.1977

Cited by 32 publications

(25 citation statements)

References 24 publications

(35 reference statements)

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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: 96%

“…In previously reported studies of monoclonal anti-CT antibodies, all stable clones of hybridomas isolated were characterized. In contrast, the 17 hybridoma cultures on the basis of preliminary screening tests. This approach facilitated our recognition of new specificity patterns but probably biased the relative frequencies with which the different specificity groups of monoclonal antibodies were represented in our collection.…”

Section: Resultsmentioning

confidence: 99%

“…Based on classical serologic studies, Hejtmancik et al (17) and Markel et al (26) proposed that CT-A and CT-B share one or more epitopes. Remmers et al (31) isolated two hybridoma cultures that produced antibodies which reacted both with CT-A and CT-B in solid-phase radioimmunoassays, but the specificity of the crossreactions observed in the radioimmunoassays was not confirmed by an independent method such as the Western blot technique.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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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“…Cholera toxin (lot no. 0175) was purified from fermentors inoculated with Vibrio cholerae Inaba 569B as previously described (10,16). Subunit purification.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Antigenic Determinants of Cholera Toxin Subunits

et al. 1979

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The antigenic specificity of purified preparations of A subunit, B subunit, a chain, and y chain of cholera toxin was studied by double immunodiffusion and radioimmunoassay with antisera produced in rabbits and mice. Rabbits immunized with A subunit produced serum antibodies which were capable of binding radiolabeled A subunit, a chain, and B subunit. Rabbits immunized with a chain produced serum antibodies that would bind radiolabeled a chain and A subunit. Rabbits immunized with the B subunit produced serum antibodies monospecific for the B subunit. The y chain did not elicit measurable antibodies in rabbits or mice as evidenced by radioimmunoassay or double immunodiffusion. A sensitive competitive radioimmunoassay was developed in which the B subunit could inhibit binding ofradiolabeled A subunit and a chain with either antisera prepared against A subunit or a chain. Neither the A subunit nor the a chain could inhibit binding of B subunit with the antiserum prepared against B subunit. In addition, selected elution fractions obtained during A-and B-subunit purification were used to immunize groups of mice. Mice responded to immunization with the elution fractions of A subunit by producing anti-A-subunit and anti-B-subunit serum antibody responses, whereas mice immunized with elution fractions of B subunit produced only antibodies specific for the B subunit. An equimolar amount of the two resulting protein peaks was used to immunize two groups of rabbits. Rabbits immunized with A subunit, produced a serum anti-B subunit response equal to that of rabbits immunized with the B subunit. Rabbits immunized with equimolar concentrations of A and B subunits were observed to be equally protected against intestinal loop challenge with Vibrio cholerae Inaba V86. The A subunit, not the B subunit, was biologically active when tested by the S49 mouse lymphosarcoma cell test. These studies provide additional evidence supporting the hypothesis that the A subunit, specifically of a chain, of cholera toxin contains antigenic determinants in common with the B subunit. lactosyl ceramide) cell membrane receptors (12, 615 on August 5, 2020 by guest http://iai.asm.org/ Downloaded from

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“…2 (12) 22.1 f 0.6 (22) 19.2 f 0.6 (19) 22.7 f 0.8 (23) 18.3 2 0 . 8 (18) 9.5 f 0.2 (10) 2.3 f 0.04 (3) 9.5 f 0.2 (10) 15.0 f 0.6 (15) 16.1 50.4 (16)…”

Section: :Tsmentioning

confidence: 99%

Structure, function, and antigenicity of cholera toxin

Markel¹,

Hejtmancik²,

Peterson³

et al. 1979

J Supramol Struct

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Chemical modification of intact cholera toxin or its B subunit by either partial nitration or reduction and alkylation did not result in significant loss of biological activity as determined by measurement of cyclic AMP in Chinese hamster ovary cells. Complete nitration or succinylation in the presence of guanidine hydrochloride resulted in complete loss of biological activity and significantly affected the immunoreactivity of the toxin and B subunit. Compositional analyses of both the isolated alpha and gamma chains of the toxin were typical of globular proteins and did not reveal significant hydrophobicity. Analysis of antigenic relationships by radioimmunoassay indicated a partial crossreactivity between the alpha chain and the B subunit of cholera toxin. Since previous structural studies of the beta chain of cholera toxin indicated chemical similarity with the glycoprotein hormones [Kurosky et al. Science 195:299 (1977)], radioimmunoassay procedures were employed to investigate for possible crossreactivity. No evidence of crossreactivity between cholera toxin subunits and subunits of ovine luteinizing hormone was found.

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Radioimmunoassay for the Antigenic Determinants of Cholera Toxin and Its Components

Cited by 32 publications

References 24 publications

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

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

Characterization of the Antigenic Determinants of Cholera Toxin Subunits

Structure, function, and antigenicity of cholera toxin

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