The performance of Fab-fragments generated from anti-SA MAb in the protein L assay.

<p>Fab-fragments were diluted from 200 nM to 50 nM, and a dilution series (50 nM to 12.5 nM) of anti-SA MAb acting as control was tested in parallel. The third line represents a control for fluorescence background induced by AF-labeled protein L and Eu-SA. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Eu- and AF-labeled protein L titration assays.

<p>AF-labeled protein L was titrated (100–6.3 nM) separately with <b>A</b>) Eu-labeled GST-VP1u and <b>B</b>) Eu-labeled SA. Eu-labeled protein L was titrated (100–6.3 nM) separately with <b>C</b>) AF-labeled SA and <b>D</b>) AF-labeled GST-VP1u. The anti-GST antibody was used as control for SA and anti-SA antibody for GST-VP1u. The third line represents a background control without antibody. The y-axis represents response counts obtained from Victor fluorometer. The error bars represent ± standard deviation between parallel wells.</p

TR-FRET signals induced by anti-SA antibody diluted in human IgG at various concentrations.

<p>Anti-SA Antibody was diluted in human IgG 1/4, 1/8 and 1/16, and tested with two dilutions of AF-labeled protein L: 100 nM and 200 nM, while the antigen concentration was kept constant (10 nM). Dilutions of anti-human IgG (80 nM), anti-SA (20 nM) were used as antibody controls, and buffer with AF-L and Eu-SA at 10 nM was used as background control. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Antibody titration assays using AF-labeled or Eu-labeled protein L.

<p><b>A</b>) Anti-GST antibody titrated against Eu-labeled GST-VP1u and AF-labeled protein L. <b>B</b>) Anti-SA antibody titrated against Eu-labeled SA and AF-labeled protein L. <b>C</b>) Anti-SA antibody titrated against AF-labeled SA and Eu-labeled protein L. <b>D</b>) Anti-GST antibody titrated against AF-labeled GST-VP1u and Eu-labeled protein L. In all setups antibody concentrations were from 3.1 nM to 50 nM, and the antigen and protein concentration was constant (20 nM). Anti-GST antibody was used as a control for SA assays, and anti-SA for GST assays. The third line represents a background control with no antibody. The y-axis represents response counts obtained from Victor² fluorometer. The error bars represent ± standard deviation between parallel wells.</p

Purification of IgM and IgY using gel permeation chromatography.

<p><b>A)</b> The IgM and IgY containing pellet from the PEG precipitation was loaded onto a 10/30 Superdex 200HR column (GE Healthcare) and the proteins were eluted with PBS at a flow rate of 0.4 ml/min. Dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection. <b>B)</b> Fractions collected during the gel permeation chromatography were analysed by SDS-PAGE under non-reducing conditions, the left lane shows molecular weight marker (Precision Plus Protein Dual Color, Bio-Rad) and the subsequent lanes labelled f1-f17 represent the fractions (marked with * in the chromatogram). <b>C)</b> The fractions (the left “peak” in A, corresponding to f1-f9 in B) containing IgM were pooled, concentrated using a 100 kDa centrifugal filter (Millipore), and loaded onto a 16/60 Sephacryl S-200HR column (GE Healthcare). The proteins were eluted with PBS at flow rate of 1.0 ml/min, and dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection. <b>D)</b> The fractions (the second “peak” in A, corresponding to f10-f17 in B) containing IgY were pooled, concentrated using a 100 kDa centrifugal filter (Millipore), and loaded onto a 16/60 Sephacryl S-200HR column (GE Healthcare). The proteins were eluted with PBS at a flow rate of 1.0 ml/min, and dual wave length absorbance monitoring, A_260nm and A_280nm, was used for protein detection.</p

Antibody titration in ELISA.

<p>Monoclonal <b>A</b>) SA and <b>B</b>) GST antibodies were titrated in SA- or GST coated ELISA. The error bars represent ± standard deviation between parallel wells.</p

SDS-PAGE analysis of the purified IgM and IgY.

<p>The IgM and IgY containing fractions were concentrated using a 100 kDa cut off centrifugal filter (Millipore), and the protein composition determined by SDS-PAGE. The left panel shows proteins separated and visualised using Coomassie staining on a 5% SDS-PAGE under non-reducing conditions, the right panel shows proteins separated on a 8% SDS-PAGE under reducing conditions. The left lane in both gels shows the molecular weight marker (Precision Plus Protein Dual Color, Bio-Rad).</p

Detection of anti-UHV NP antibodies in snakes with BIBD.

<p>The sera were collected from <i>Boa constrictors</i> with BIBD as characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158417#pone.0158417.ref020" target="_blank">20</a>].</p

ADAM8 interacts with TOCA1 and SNX33 in human cells.

<p>SNX33 and TOCA1 were expressed as EGFP-fusion proteins by transiently transfected 293T cells alone or with HA-tagged ADAM8 as indicated on top of the figure. The presence of ADAM8 itself (top panel) or TOCA1 or SNX33 that was associated with it (second panel from top) in anti-HA immunoprecipitates (IP) from the lysates of these cells was examined by Western blotting. Similar total levels of ADAM8, TOCA1, and SNX33 between the transfected cells were confirmed by a Western blotting analysis of the unselected lysates (Lysate). Blotting for the endogenous α-tubulin in these lysates was included as an additional loading control.</p

Screening of sera from <i>Boa constrictors</i> with BIBD for antibodies against reptarenaviruses by immunoblotting.

<p>Protein pools of Ni-NTA affinity purified recombinant UHV NP (the full-length protein, and N- and C-terminal fragments) separated by SDS-PAGE under reducing conditions were used as the antigens. The membranes were probed with snake serum, and detection of binding was done using either anti-IgY or anti-IgM in combination with an IRDye800 labelled secondary antibody. The immunoblot signals were recorded with Odyssey infrared imaging system (LI-COR Biosciences). The snake serum (or blood) samples #1–4 to #6–9 correspond to the animal numbers of our earlier study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158417#pone.0158417.t001" target="_blank">Table 1</a>, and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158417#pone.0158417.ref020" target="_blank">20</a>]). The left lanes show the molecular weight marker (Precision Plus Protein Dual Color, Bio-Rad). The arrows indicate the size of the recombinant proteins.</p