To quantify the baseline levels of anti-influenza heterosubtypic Abs in human serum, we used IVIG as representative of pre-immune IgG Ab composition in the general population, which includes individuals who are likely exposed to seasonal Figure 2 . Heterosubtypic antibodies against influenza A viruses in intravenous immunoglobulin (IVIG). H5-VN04 was immobilized on magnetic beads (H5-beads) and the beads were used to affinity purify antibodies (Abs) from the IVIG sample. F10-like Abs and the remaining H5-bound Abs were purified separately. F10-like Abs were purified by Bio-F10 competition elution followed by multiple steps of streptavidin-beads absorption to eliminate Bio-F10 completely. The remaining H5-bound Abs on the H5 beads were eluted with standard acid elution method followed by a complete absorption of Bio-F10 using Streptavidin-beads as well.An enzyme-linked immunosorbent assay (ELISA) detecting Bio-F10 was used to confirm that no residual Bio-F10 remained in either Bio-F10-eluted or the acid-eluted samples. The binding activity of these purified Abs to H1-NY18 (A), H5-VN04 (B), H3-A2/68 (C), and H7-NL03 (D) was measured by ELISA at different serially diluted concentration. The neutralization activity of these samples was measured using neutralization assay with pseudotyped viruses of H1-1918 (E) and H5-TH04 (F); 80R was used as a negative control mAb that is specifically against the spike protein of severe acute respiratory syndrome coronavirus [24] . OD 450, optical density at 450 nm.