Characterization of the binding of cholera toxin to ganglioside GM1 immobilized onto microtitre plates

✍ Scribed by R. M. Dawson

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 337 KB
• Volume: 25
• Category: Article
• ISSN: 0260-437X
• DOI: 10.1002/jat.1015

✦ Synopsis

Ganglioside G M1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to G M1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (G M1 -ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for G M1 (methanol or phosphate-buffered saline), the pH of aqueous solvents (7.4-10.2) and the temperature (4-37 °C). High and nearmaximal rates of absorbance change in the assay were found for lower concentrations of G M1 (100 ng ml - ----1 ) and for shorter incubation times (a few hours) than reported in the literature. A method was devised to provide a semi-quantitative estimate of the amount of G M1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized G M1 required ≥ ≥ ≥ ≥ ≥1.5 h for maximal assay results. The failure of free G M1 in solution to displace cholera toxin once bound to immobilized G M1 indicated that binding to immobilized G M1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin-G M1 complex. Copyright