FIGURE 2.
The anti‐αGal antibody binds most pneumococcal serotypes. Flow cytometry analyses showing antibody reactivity with various bacteria. (A) Example plot showing antibody reactivity with serotype 9V pneumococci after incubation with buffer only, negative control IgG (IgG anti‐CD20, i.e. irrelevant specificity) at 10 mg/L, purified human anti‐αGal at 5 mg/L or normal human IgG (nhIgG) at 500 mg/L. NhIgG is expected to contain antibodies to pneumococci and served as positive control. IgG on the pneumococci was assayed with fluorescently labelled rabbit F(ab’)2 anti‐human IgG. ‘FI’: Fluorescence intensity. (B) Pie‐chart summarizing reactivity of anti‐αGal (5 mg/L) with 91 serotypes of S. pneumoniae. (C) Level of reactivity with the reactive pneumococcal serotypes and five control bacterial strains. Control strains were E. coli O86 (EcO86) and four unencapsulated pneumococcal strains: C‐mutant (covered by a thick layer of cell wall (CW) polysaccharide), Rough1, Rough2 and Rough4. The columns are the mean reactivity (MFIrel) and standard deviation of two separate experiments. MFIrel was the MFI in experiments with anti‐αGal relative to the MFI in the same experiment but without primary antibody (fluorescently labelled anti‐hIgG was present in both experiments). A positive antibody reaction was defined as a mean MFIrel at least two standard deviations above 1·10 (corresponding to the highest reactivity observed in parallel and equal experiments but with the negative control IgG anti‐CD20 as primary antibody). The figure shows that anti‐αGal reacts with numerous serotypes although they do not possess terminal Galα3Gal in their polysaccharide structure (red columns) (see also Figure 3). Several serotypes demonstrate higher reactivity than the positive control strain, EcO86. The serotypes highlighted in yellow were selected for further experiments