Figure 5

ELISA control experiments. A. Spiking with cholesterol at the end of the growth period does not alter Lewis antigen expression. Cultures of H. pylori were grown overnight in defined medium without (control) or with 50 μg/ml cholesterol (cholesterol grown). A third flask (cholesterol spiked) was grown in the absence of cholesterol, chilled on ice, and an equivalent amount of cholesterol was added before the cells were harvested. Lewis antigens were quantitated in duplicate by whole-cell ELISA, loading 300 ng cellular protein per well. Ratios for plus:minus cholesterol were calculated from average net absorbance readings in each assay, and the plot displays mean ratios ± sem for three to five independent ELISA runs. P values were calculated in two-tailed Student t-tests for the null hypothesis that the ratio equals 1. For comparisons labeled ns, P > .05. B. Equivalent binding of cells to ELISA plates. Samples of H. pylori that were grown in parallel cultures in the absence (white bars) or presence of 50 μg/ml cholesterol (grey bars) were applied to multiwell plates in the same manner as for Lewis antigen ELISA assays, adding 500 ng of cellular protein per well. Following overnight attachment, wells were washed twice with Dulbecco's phosphate-buffered saline, then protein in adherent cells was quantitated using the BCA reagent. Mean values ± sd of quadruplicate wells are shown.