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Figure 2.

Figure 2. From: Wide Prevalence of Heterosubtypic Broadly Neutralizing Human Anti-Influenza A Antibodies.

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.

Jianhua Sui, et al. Clin Infect Dis. 2011 April 15;52(8):1003-1009.
Figure 1.

Figure 1. From: Wide Prevalence of Heterosubtypic Broadly Neutralizing Human Anti-Influenza A Antibodies.

Serological study of 77 paired pre- and post-immune serum samples from H5N1 vaccinees. A–D, Enzyme-linked immunosorbent assays (ELISAs). Serum samples were serially diluted and applied to H5-VN04– (A and B), H3-A2/68– (C), or H7-NL03– (D) coated ELISA plates and the binding of immunoglobulin (Ig) M or IgG in serum samples to hemagglutinin (HA) proteins were determined using secondary Ab HRP-anti-human IgM (A) or IgG (B - –D). The binding levels are shown as optical density at 450 nm (OD 450). E, microneutralization assay (MN) titer against H5N1 virus; y-axis shows the Log2 MN titer. F, Competition ELISA. Pre- and post-immune serum samples from H5N1 vaccinees were tested for their competition activity against a Group 1-specific BnAb, F10, binding to H5-VN04. Serially diluted serum samples were mixed with 3 ng/mL Bio-F10 and applied to H5-coated ELISA plates. The serum competition for binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin. The serum competition activity is shown as percentage of inhibition. For all panels except panel E, data at 1 representative serum dilution are shown, as follows: panel A, 1:270; panel B, 1:5120; panels C and D, 1:2430; and panel F, 1:90. For all panels, data are shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles, respectively. The dots above and below the whiskers are data points beyond the 95th and 5th percentiles.

Jianhua Sui, et al. Clin Infect Dis. 2011 April 15;52(8):1003-1009.

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