Anti-cholera Toxin Antibody Research Articles

A lavage technique allowing repeated measurement of IgA antibody in mouse intestinal secretions

Mouse intestinal secretions can be readily obtained without harm to the mice by administering a lavage solution to them intragastrically followed by pilocarpine intraperitoneally. These secretions are rich in proteases but this enzyme activity can be blocked by addition of a mixture of inhibitors. Both total and specific IgA antibody could be measured in these secretions using ELISA techniques. The total IgA recovered was found to vary considerably, even in the same group of mice sampled on multiple occasions. Specific IgA anti-cholera toxin antibody was easily demonstrable in the intestinal secretions of mice fed cholera toxin but not of mice fed an irrelevant antigen. Expression of the specific IgA antibody per unit of total IgA recovered is desirable in order to correct for the variable recovery of IgA.

Immunocytochemical study on cholera toxin binding sites by monoclonal anti-cholera toxin antibody in neuronal tissue culture

An indirect method of immunocytochemistry showed that cholera toxin and its B-subunit served as specific neuronal surface markers in conjunction with monoclonal anti-cholera toxin and FITC labeled anti-mouse Fab. The cell types which get specifically stained in culture were peripheral neurons from dorsal root ganglion cells, superior cervical ganglion cells, and cerebral neurons, all of which were rat tissue, and NGF-treated PC12 cells. Non-neuronal cells, i.e. Schwann cells, fibroblasts and glia cells, were not stained. This method can, therefore, be used to distinguish neurons from non-neuronal cells in neuronal tissue cultures, as in the case of tetanus toxin described in the literature 5,10,23,25,28,29.

Induction by cholera toxin of synchronous divisions in vivo in the epidermis resulting in hyperplasia.

Intracutaneous injection of cholera toxin, exotoxin of Vibrio cholerae, into the dorsal skin of mice, rats, and hamsters at doses of greater than 0.1 ng evoked an acute reaction at the site of injection, which was characterized histologically by an edematous reaction in the dermis and mitotic stimulation in the epidermis. Mitotic and labeling induces of basal cells of the mouse epidermis showed two peaks at 24 and 48 hr after injection, thereby producing epidermal hyperplasia. The thickness of the intrafollicular epidermis increased progressively from 32 hr after toxin injection, being greatest on day 4 and decreasing to normal on day 7. The epidermis on day 4 after injection of 1.0 ng of toxin was about 4- to 6-fold thicker than normal or phosphate buffer-treated control skin. This sequence of events indicated that cholera toxin induced two successive synchronous divisions of the epidermal cells and produced temporary hyperplasia without interfering with epidermal differentiation. The complete structure and function of the cholera toxin are required for induction of epidermal hyperplasia: no mitotic stimulation was induced by injection of the A and B units of the cholera toxin molecule or by preincubation of the toxin with anti-cholera toxin antibody and with the membrane receptor, GM1 ganglioside. Five other agents known to increase the level of intracellular cyclic AMP by different means (dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, isoproterenol, and prostaglandin E1) did not produce a skin reaction.

Identification of cholera toxin binding glycoproteins in rat intestinal microvillus membranes.

Glycoproteins of the microvillus membranes from rat small intestinal epithelial cells were examined for their ability to bind cholera toxin. Membrane glycoproteins prepared by Ricinus communis agglutinin-Sepharose affinity chromatography, which were free of glycolipids, were shown to be able to form complexes with iodinated cholera toxin by gel filtration column chromatography. These glycoproteins were further characterized by subjecting the complex of tritiated cholera toxin receptors, cholera toxin, and anti-cholera toxin antibody to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography and by localizing cholera toxin binding glycoproteins on sodium dodecyl sulfate-gels using iodinated cholera toxin. In addition to glycolipids, at least 5 glycoproteins with molecular weights of 69,000, 90,000, 100,000, 114,000, and 132,000 were identified by both methods. These results suggest that microvillus membranes contain glycoprotein and glycolipid receptors for cholera toxin.

Involvement of arginine residues in the binding site of cholera toxin subunit B

Modification of one or two of three guanido groups in the binding subunit (B) of cholera toxin was achieved at pH 9 with cyclohexanedione at 50 mM or 150 mM concentration, respectively. No change in the helix content or the pentameric structure was observed in the process. The ability to form precipitate with ganglioside G ml or anti-cholera toxin antibody was abolished only when two of the three arginine residues were modified. Analyses of Arg-containing peptides revealed that reaction with cyclohexanedione occurred first with Arg-73 and then with Arg-35. This suggests that Arg-35 or the region in proximity is involved in the interaction of subunit B with ganglioside G ml or the antibody. A diagram of secondary structure as predicted by the Chou-Fasman rule indicates that this region is within a long stretch of β-sheet configuration.

Monospecific Equine Antiserum Against Cholera Exo-Enterotoxin

An antiserum specific for Vibrio cholerae exo-enterotoxin was produced by immunization of a horse with purified choleragenoid, a natural cholera toxoid. The serum has a high titer against the toxin antigen in passive hemagglutination tests and a respectable antipermeability factor activity. It also passively protected against choleragen-induced mouse foot edema. The serum was found to be useful for assaying toxin antigen in crude and refined products by in vitro tests such as radial immunodiffusion, Lf, and quantitative precipitin titrations. Based upon experimental observations, the serum was defined as containing 1,000 flocculating units of anticholera toxin antibody per ml. A flocculating dose, or Lf, of choleragen approximates 1 mug, and that of choleragenoid is 0.625 mug. Formalin toxoids behaved like the parent toxin in these tests. The serum contains approximately 2.2 mg of antibody protein per ml, which appears to be largely, if not entirely, of the Ig(T) type. It is suggested that this serum, which is available in considerable supply, be considered for use as a reference cholera antitoxin. The horse developed symptoms of anaphylactic shock during immunization, suggesting the need for caution in projected studies on toxoid-induced immunity in man.