Sialic acids expressed by SAP are resistant to hydrolysis by the viral NA.

<p>(<b>A</b>) Wells coated with 1 µg/ml of HKx31 were incubated with increasing concentrations of fetuin or asialofetuin (ASF) for 1 hr before addition of 5 µg/ml biotin-labelled SAP. Binding of biotin-labelled SAP to HKx31 was then determined by ELISA. All incubations and washes were performed at 4°C using chilled buffers to inhibit the enzymatic activity of the viral NA. Data show a single titration (left panel) as well as triplicate wells at a specific concentration of fetuin/ASF (right panel). The absorbance of biotin-labelled SAP binding in the absence of fetuin/ASF was 1.558 (±0.105). ***p<0.001, Student’s t-test for equivalent concentrations of fetuin compared to ASF. (<b>B</b>) Purified PTX3 and SAP were subjected to 12% SDS-PAGE under reducing conditions followed by lectin blot using DIG-conjugated MAA (panel i) or SNA (panel ii) for detection of α(2,3)- or α(2,6)-linked SA, respectively. Fetuin was included as a positive control for each SA linkage. (<b>C</b>) SAP (2.5 µg) was incubated for 1 hr at 37°C with sialidase from <i>V. cholerae</i> (25 mU), HKx31 (1/20 dilution of allantoic fluid) or with buffer alone and then analysed by SDS-PAGE and DIG-lectin blot using DIG-SNA (panel i). Ponceau S stain confirmed similar amounts of SAP were transferred to the PVDF membrane prior to the lectin blot (panel ii). (<b>D</b>) ELISA plates coated with (i) fetuin (20 µg/ml), or (ii) SAP (40 µg/ml) were incubated with HKx31 (1/10 dilution of allanotic fluid) or sialidase from <i>V. cholerae</i> (0.5 mU) for 30 min at 37°C and removal of SA was determined using NA assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059623#s2" target="_blank">Material and Methods</a>. Uninfected allantoic fluid (AF) was included as a negative control. Data shows the mean absorbance (±1 SD) of triplicate wells and is representative of at least two independent experiments. ***p<0.001, One-way ANOVA compared to uninfected allantoic fluid control or buffer alone control.