Inhibition of phagocytosis by IVIg is not enhanced in fractions enriched for sialic acid. (A) Inhibition of phagocytosis by IVIg enriched or depleted for sialic acid by SNA fractionation. Means and standard error of the mean of at least 4 experiments for each concentration are shown. (B) Inhibition of phagocytosis by IVIg treated with neuraminidase to remove sialic acid residues compared with sham-treated IVIg. The level of sialylation of IVIg was determined by mass spectrometry as in panel C, the results of which are indicated by percentages in the figure. Means and standard error of the mean of 4 experiments for each concentration are shown. (C) Example of mass spectrometry spectra, in this case showing trypsin-generated glycopeptides of anti-trinitrophenol IgG1 produced in the absence (IgG1 Fc sialylation-negative) or presence (IgG1 Fc sialylation-positive) of galactosyl- and sialyltransferase. Percentage Fc-sialylation is indicated. A minority of the peaks appear to belong to glycopeptides with a modified peptide mass and are labeled accordingly: *mass increase of +218.1 Da, **mass increase of +436.2 = 2 × 218.1 Da. Pep, peptide; green circle, mannose; yellow circle, galactose; blue square, N-acetylglucosamine; red triangle, fucose; purple diamond, N-acetylneuraminic acid (sialic acid). (D) Inhibition of phagocytosis by recombinant human IgG1 from panel C with very low or enhanced Fc-sialylation. Means and standard error of the mean of at least 5 experiments for each concentration are shown. P values in this figure were determined using 2-way ANOVA.