A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.

Ganglioside GM 1 ELISA. Ninety-six-well microtiter plates were coated overnight with 50 l of 2-fold serial dilutions of ganglioside GM 1 (Supelco, Sigma-Aldrich, St. Louis, MO) in PBS, starting at 150 nM, and then blocked with 10% horse serum in PBS. All steps were performed at 37°C for 1 h, after which samples were aspirated off and wells were washed three times with PBS plus 0.05% Tween 20. Each holotoxin was assayed in triplicate by incubating 100 l of 50-ng/ml holotoxin per well (approximately 60 fmol), followed by 1/5,000 rabbit anti-cholera toxin B subunit (B10) and then 1/2,000 HRP-conjugated goat anti-rabbit serum (Thermo Fisher Scientific Inc., Rockford, IL), followed by o-phenylenediamine (OPD) substrate (Sigma-Aldrich), and the reaction was stopped with 3 M HCl. Absorbance was read at 450 nm. Y1 assay. Mouse Y1 adrenal cells (ATCC CCL-79) were grown at 37°C in a humidified 5% CO 2 atmosphere in RPMI medium with 5% fetal bovine serum with 1ϫ penicillin-streptomycin (Life Technologies), and 96-well plates were seeded with 10 4 cells per well. One-hundredmicroliter volumes of 2-fold serial dilutions of toxins were added to semiconfluent monolayers and incubated overnight, followed by scoring for toxin-induced rounding. One unit of toxin was defined as the amount contained in the last dilution giving 75 to 100% rounding of cells.