NTHi uptake and expression of Neu5Gc from baby food and its recognition by purified human anti-Neu5Gc antibodies. (A) WT NTHi strain 2019 (WT, solid line) and Sia transporter mutant strain of 2019 (SiaT Mutant, dashed line) were grown in Sia-free media with or without the addition of commercially available baby foods and analyzed by flow cytometry for Neu5Gc staining using the chicken anti-Neu5Gc IgY antibody. Secondary antibody for WT and SiaT Mutant (not depicted) and IgY control for SiaT Mutant (not depicted) all showed similar shifts compared with the IgY control for WT (solid gray). (B) Anti-Neu5Gc IgG antibodies purified from serum from a single individual (S34) was used to probe NTHi in a whole-cell ELISA. NTHi was grown in Sia-free media (Sia-Free NTHi) or Sia-free media with 1 mM Neu5Gc (Gc-NTHi), and binding of human antibodies is shown the mean of triplicate values at OD₄₀₅. Error bars represent SD. Statistical analysis was performed using an unpaired two-tailed Student’s t test. ***, P < 0.0001. Data are representative of two (A) and three (B) independent experiments.