Assay Cholera toxin

Rowe-Taitt C.A., Cras J.J., Patterson C.H., Golden J.P., Ligler F.S., A ganglioside-based assay for cholera toxin using an array biosensor, Anal. Biochem. 2000a 281 123-133. 

V. cholerae B subunits of the cholera toxin (CTB) Tobacco leaf chloroplast No immunogenicity assays performed. 119... 

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

Purified CTA or LTA is required for the assays described herein and can be generated and purified from CT or LT holotoxin using methods originally described for CT. Gel filtration in 0.1 M glycine buffer (pH 3.2) containing 6 M urea (Ohtomo ef al., 1976) or 5 % formic acid (Lai et al., 1976) results in separation of the A subunit from B subunit monomers. More recently, a reverse-phase HPLC procedure has been described for cholera toxin subunit purification (Pearson etal., 1986). Like CT, purified CTA can be purchased from several sources (see Section 2.2.3). 

Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin. Pseudomonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C. botulinum C2 toxin (Aktories and Just, 1993)), C3 is a mono-ADP-ribosyltransferase (Aktories et ai, 1988b). Treatment of ADP-ribosylated Rho with phosphodiesterase releases 5 -AMP and not phosphoribosyl-AIVtP, a cleavage product of poly(ADP-ribose) (Aktories et ai, 1988b Rubin ef a/., 1988). Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADP-ribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions. 

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