Competition assays to measure cross-reactivity of anti-GAD65 (a) and anti-GAD67 (b).

<p>Results, shown as mean and range of duplicates, of inhibition assay for three representative sera inhibited with GAD65 and GAD67. Reactivity with ¹²⁵I-GAD65 or ¹²⁵I-GAD67 (dark grey bars) was inhibited with 100-fold excess of the same GAD (homologous inhibition, black bars). In contrast, unlabelled GAD65 substantially or completely inhibited anti-GAD67, although unlabelled GAD67 did not inhibit anti-GAD65 reactivity (heterologous inhibition, light grey bars).</p

Results of heterologous inhibition of anti-GAD67 with GAD65, or anti-GAD65 with GAD67 for 30 sera, and of homologous inhibition (italics) of anti-GAD65 with GAD65 and anti-GAD67 with GAD67 for 17 sera, shown as Mean TC% ± SD for each group.

<p>*not significant, dependent t-test.</p><p>**p&lt;0.0001, dependent t-test.</p

Affinity of anti-GAD65 and anti-GAD67 in five sera that contain both anti-GAD65 and anti-GAD67 and in five that contain anti-GAD65 only.

<p>Affinity of anti-GAD65 and anti-GAD67 in five sera that contain both anti-GAD65 and anti-GAD67 and in five that contain anti-GAD65 only.</p

Antibodies to GAD65 and GAD67 detected by RIP.

<p>(a) Comparison of antibody reactivity to GAD65 (x-axis) and to GAD67 (y-axis), measured as the percent of the total counts of radioactivity precipitated by each serum (%Total counts) in 85 sera that contain anti-GAD65. The horizontal dotted line indicates the upper limit of normal for anti-GAD67 and the vertical dotted line indicates the upper limit of normal for anti-GAD65, based on mean+3SD of reactivity of 10 normal controls. None of the normal controls showed any reactivity beyond these levels. (b) Titration curves for anti-GAD65 and anti-GAD67 in three representative sera that contain high levels of both anti-GAD65 and anti-GAD67. , (□) and (▴) represent three different sera samples.</p

Surface localisation of <i>P. multocida</i> PlpE by immunofluorescence assay.

<p>Bacteria were fixed with paraformaldehyde, incubated with chicken antiserum against PlpE, stained with Alexa Fluor 488 goat anti-chicken IgG, and visualized by fluorescence microscopy. Fluorescence (panels: a, c, e) and DIC (Differential Interference Contrast) (panels: b, d, f) selected images of the bacterial Z-stack cross-sections. Control staining with antiserum raised against an unrelated protein showed no surface fluorescence (data not shown). Scale bar  = 1 µm.</p

Protective capacity of 8 M urea-denatured, recombinant PlpE in chickens and mice.

a<p>Results of chicken experiment 1 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039973#pone-0039973-t001" target="_blank">Table 1</a>.</p>b<p>All animals deemed incapable of survival were euthanized in accordance with animal ethics requirements.</p>c<p>Compared to adjuvant control: Fisher’s exact test.</p

Western immunoblot analysis of PlpE expression in <i>P. multocida</i> whole cell lysates probed with chicken antiserum against recombinant PlpE.

<p>Lanes: wild type strain (lane 1); <i>plpE</i> mutant (lane 2); complemented mutant (lane 3); mutant transformed with empty vector (lane 4). Numbers on the left indicate the positions of molecular mass standards (in kDa). Arrow indicates the position of the 39 kDa PlpE. Pre-bleed serum showed no reactivity (data not shown).</p

Recombinant <i>P. multocida</i> proteins tested as vaccine candidates in chickens.

a<p>- Gene numbers are from the Pm70 strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039973#pone.0039973-May1" target="_blank">[28]</a> homolog of the X-73 genes amplified and cloned.</p>b<p>- Expression vectors: pBAD-DEST49, a Gateway® destination vector that features a thioredoxin fusion tag; pDEST-41BA a modified Gateway-adapted expression vector containing a NusA solubility tag <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039973#pone.0039973-AlHasani1" target="_blank">[20]</a> (originally derived from pLIC-NusA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039973#pone.0039973-Cabrita1" target="_blank">[48]</a>); pDEST-17, a Gateway® destination vector for production of tagless native protein.</p>c<p>- I  =  insoluble; S  =  soluble protein.</p>d<p>- SignalP server signal peptide prediction. In general PCR primers were designed so as to amplify the gene region encoding the mature length protein, excluding the signal sequence, except where a signal sequence could not be predicted; in this case the primers were designed to encompass the entire gene.</p>e<p>- immunogenic reaction against the recombinant protein.</p>f<p>- serum reacted with a protein of appropriate size in a whole cell lysate derived from <i>P. multocida</i> grown <i>in vitro</i> at 37°C.</p>g<p>- positive (X-73 whole killed cells) and negative controls used in the vaccine trial.</p>h<p>- Due to its large size, PM0714 was purified in three overlapping sub-fragments spanning the entire protein.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Conversion activity of brain derived PrP^C in the CAA seeded with infected brain homogenates is sensitive to ionic strength and inhibited by the specific depletion of heparan sulphate.

<p>(A) The CAA was performed using IBH diluted in UBH prepared from WT and KO mice in Tris-HCl pH 7.4 and the indicated concentrations of NaCl. ** Indicates a significant reduction in conversion activity relative to 125mM NaCl. B) The CAA was performed using IBH diluted in UBH prepared from WT mice in 125mM NaCl/Tris-HCl pH 7.4 after treatment with Heparinase I (H), Heparinase III (HS), Chondroitinase ABC (Ch), their corresponding buffers (underlined) or without treatment (Con). Conversion activity was determined as the fold increase in immunoreactive signal of WT relative to KO reactions after overnight incubation at 37°C and treatment with PK (100µg/ml, 1hr at 37°C). Quantification (A, B) is based on at least three experiments, mean and SEM are shown. **p&lt;0.01, ***p&lt;0.001 using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad, Prism). C) The amount of sGAG purified from UBH treated with Heparinase I (H), Heparinase III (HS) and Chondrotinase ABC (Ch) or untreated (Con) was determined by Blyscan analysis and normalised to the amount of sGAG recovered from buffer controls (not shown). D) The absorbance (254nm) of sGAG eluted from a Q-Sepharose HiTrap anion exchange column in increasing concentrations of NaCl (0–1M). GAGs were purified from control (□), Heparinase I treated (⋄) and Heparinase III treated (○) or Chondroitinase ABC treated (+) brain homogenates. Quantification (C, D) is based on an analysis performed in duplicate.</p