To isolate H5-bound Abs and F10-like Abs from intravenous immunoglobulin (IVIG; 100 mg/mL, Gamunex IVIG; Talecris Biotherapeutic), we first immobilized H5-VN04 proteins on magnetic beads (Dynabeads M-270 Epoxy; Invitrogen) in accordance with the manufacturer's manual, and then used step-wise Bio-F10 elution and acid elution to separate the F10-like Abs and H5-bound Abs present in the IVIG. Specifically, 5 3 10 8 of H5-beads were incubated with 1.5 mL of IVIG (100 mg/mL) overnight at 4°C, washed extensively with 0.1% BSA/PBS, followed by Bio-F10 elution (100 lg/ml, 0.5ml, total 50 lg) overnight at 4°C. The remaining H5-bound Abs on the H5-beads were then isolated with acid elution (pH 5 2.8; buffer, 500 lL/10 9 H5beads). Next, the Bio-F10 eluents and the acid eluents were incubated with Strep-T1 beads (250 lL of 4 3 10 8 beads; Invitrogen) at 4°C for 4-8 h each time for 2 and 3 times, respectively. ELISA was performed to verify the Bio-F10 in both samples was completely removed after Strep-T1 beads absorption. The purified H5-bound Ab and F10-like Abs, as well as unpurified IVIG, were quantified by ELISA using known concentrations of F10 monoclonal Ab (mAb) as standards. The aforementioned purification procedure was scaled up proportionally to obtain enough H5bound Abs and F10-like Abs to test their cross-reactivity against different influenza A subtypes by ELISA and pseudotyped virus neutralization assay, as described elsewhere [1] .