Characterization of the binding of cholera toxin to ganglioside G_M1 immobilized onto microtitre plates

Abstract

Ganglioside GM1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to GM1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (GM1-ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for GM1 (methanol or phosphate-buffered saline), the pH of aqueous solvents (7.4-10.2) and the temperature (4-37 degrees C). High and near-maximal rates of absorbance change in the assay were found for lower concentrations of GM1 (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 GM1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized GM1 required > or =1.5 h for maximal assay results. The failure of free GM1 in solution to displace cholera toxin once bound to immobilized GM1 indicated that binding to immobilized GM1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin-GM1 complex.

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

“…The relationship between Ctx-HRP concentration and peroxide-induced colour formation is sigmoidal, a result in agreement with other studies. 49,50 Furthermore, the concentration of Ctx-HRP corresponding to half-maximal binding was estimated to be 15 ng mL À1 or 0.153 nM. Other important developments include the incubation period between potential inhibitors and Ctx, which was set at 2 h before addition to the plate to maximise interactions between sugar and toxin.…”

“…A further 2-h incubation 'on plate' was chosen because time-dependency experiments have shown that the maximum binding of Ctx to GM1 is reached in 90 min. 49 Bound/uninhibited Ctx-HRP was developed with 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) rather than o-phenylenediamine free base as the peroxidase substrate because of its sensitivity in detecting low concentrations of Ctx-HRP. The statistical mean of each set of absorbance readings was used to calculate the concentration of Ctx-HRP that was not inhibited, and consequently able to bind to the immobilised GM1 surface.…”

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

“…The assembled pentamer is capable of binding five ganglioside GM1 receptors at once (Merritt et al 1994;, 1998. The CTB binds very tightly to GM1, with reported Kd values in the range of 5 pM to 1 nM based on surface plasmon resonance and oncell binding measurements (Cuatrecasas 1973;Kuziemko et al 1996;MacKenzie et al 1997;Dawson 2005).…”

Silica supported phospholipid layers doped with GM1: A comparison between different methods

“…Several methods for supported lipid layer formation onto hydrophilic surfaces are described in the literature, namely, Langmuir-Blodgett transfer [8,9], spin coating [16], vesicle fusion [3,4,8,[10][11][12]17], and deposition from mixed micelles [5,6,13]. The methods to form the lipid layers selected in this study were vesicle fusion, since it was found to be the most common method, adsorption of mixed micelles, due to the fact that little work has been devoted to their use, and the so called hydration method (deposition from solvent), which is normally employed in the ELISA assays [18,19]. A comparison between different methods of preparing phospholipid surfaces was reported by Malmsten [20].…”