Comparison of binding activities of S139/1 IgA and IgG antibodies.

<p>Binding activities of S139/1 IgA (continuous lines) and IgG (dashed lines) were tested in ELISA. Disrupted viral particles of Aichi/H3, WSN/H1, Adachi/H2, and Maryland/H13 were used as antigens. Data are mean values of duplicate wells. EC₅₀ values calculated based on the ELISA data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085582#pone-0085582-t001" target="_blank">Table 1</a>.</p

Comparison of binding abilities of S139/1 IgA and IgG to influenza A viruses.

<p>The concentrations of MAbs giving 50% of maximal binding to influenza A viruses were determined according to regression curves obtained from ELISA. The assays were performed three times and the representative data are shown.</p

SDS-PAGE analysis of purified S139/1 IgA and IgG antibodies.

<p>Equal amounts (2.5 µg) of purified S139/1 IgA and IgG antibodies were analyzed by SDS-PAGE under reducing conditions (5%–20% gradient gel) (A). Polymeric forms of purified S139/1 IgA and IgG antibodies (5 µg) were analyzed under nonreducing conditions (3%–10% gradient gel) (B).</p

Viral release from infected cells cultured in the presence of S139/1 IgA or IgG antibodies.

<p>After inoculation with Aichi/H3 (A and B), WSN/H1, Adachi/H2, or Maryland/H13 (C and D), MDCK cells were cultured in the presence of S139/1 IgA or IgG antibodies at the concentrations of 1.0 or 0.1 µg/ml (A and B) and 1.0 µg/ml (C and D). Supernatants were collected 6 and 12 hours after infection, and viral proteins of influenza viruses released into the supernatants were detected by Western blotting (B and D). The relative quantity of the M1 protein was calculated based on the band intensity by using Image Lab version 3.0 (Bio-Rad) (A and C). The intensity of the M1 protein bands detected in the control supernatants collected from infected cells cultured without a MAb (w/o MAb) was set to 100%. Experiments were performed 3 times, and averages and standard deviations are shown (A and C). Statistical significance was analyzed by Student's t-test (**p<0.01, *p<0.05). Asterisks placed directly above the bars indicate significant differences compared to respective controls, and asterisks placed between the bars show significant differences between S139/1 IgA and IgG antibodies.</p

Comparison of neutralizing activities of S139/1 IgA and IgG antibodies.

<p>Appropriately diluted viruses were mixed with S139/1 IgA (continuous lines) or IgG (dashed lines) at the indicated dilutions. Neutralizing activities were evaluated by counting the number of plaques formed on MDCK cells. IC₅₀ values calculated based on the neutralization curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085582#pone-0085582-t003" target="_blank">Table 3</a>.</p

Viral particles deposited on the surface of infected cells cultured with S139/1 IgA.

<p>MDCK cells were infected with Aichi/H3 and incubated for 8 hours in the presence of S139/1 IgA (A and D), IgG (B and E), or in the absence of antibodies (C and F). TEM images of the cell surface are shown at high (A to C) and low (D to F) magnifications. Scale bars represent 1 µm (A to C) and 2 µm (D to F).</p

Neutralizing activities of the samples of mice immunized with the H9N2 virus in a standard plaque-reduction test.

<p>Appropriately diluted viruses were mixed at indicated dilutions with the serum (A), TW (B), or NW (C) sample of mice immunized with H9N2 intranasally (i.n.) or subcutaneously (s.c.). Neutralizing activities were evaluated by counting the number of plaques formed on MDCK cells.</p

Heterosubtypic plaque reduction interfered by anti-mouse IgA antibodies.

<p>NW samples of mice immunized with H9N2 virus intranasally (i.n.) were pretreated with anti-mouse IgA or anti-mouse IgG antiserum (1 µg/ml). MDCK cells infected with H12N5 virus were incubated in the presence or absence of the NW samples treated or nontreated with the antiserum.</p

Detection of viral proteins in the supernatants of cells infected with H9N2, H12N5, or H5N1.

<p>MDCK cells were infected with viruses and cultured with the TW samples of intranasally (i.n.) or subcutaneously (s.c.) immunized mice as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071534#pone-0071534-g005" target="_blank">Figure 5</a>. Supernatants were collected 7 hours after infection, and virus particles released into the supernatants was detected by Western blotting (A). The relative intensity of the HA bands compared to each of the control (PBS i.n. and s.c.) samples was obtained using Image Lab version 3.0 (BIO RAD) (B). Experiments were performed 3 times, and averages and standard deviations are shown. Student t-test was used for statistical analysis.</p

Reduced plaque formation in the presence of antibodies of mice immunized with H9N2.

<p>After being inoculated with H9N2, H12N5, H5N1, or H3N2, MDCK cells were cultured in the presence of antibodies in the serum (1∶200) (A), TW (1∶5) (B), or NW (1∶5) (C) samples of mice immunized intranasally (i.n.) or subcutaneously (s.c.). Numbers of plaques were counted and plaque reduction was calculated relative to the number of plaques formed in the presence of the samples of mice given PBS intranasally or subcutaneously for i.n. or s.c. immunized mice, respectively (D).</p