Protective effect of 2M Glucose on the ability of IgE to bind to the high affinity receptor after heating at 56°C for 30 min.

<p>RS ATL8 cells seeded at a density of 50.000 cells per well were sensitised with a 50-fold dilution of Par j 2 serum pool, which had been left unheated or heated either in the presence or in the absence of 2M Glucose. After overnight incubation with the sera, cells were stimulated with optimal concentrations of anti-IgE (1 µg/mL) or Par j 2 recombinant allergen (100 pg/mL) and luciferase production measured after 4 hours. ; *: p<0.05; **: <i>p</i><0.01; ***: p<0.001; n.s.: not significant (Student t-test).</p

Western blot demonstrating antigenicity of the <i>S. mansoni</i> proteins expressed in the WGL system.

<p>Non-purified wheat germ extracts containing <i>in vitro</i> translated IPSE/alpha-1, SmTAL1 or SmTAL2 were separated in a 4–20% SDS-PAGE gel and blotted onto NCM. Separate strips of NCM were treated with anti-TAL1 rabbit serum, anti-TAL2 rabbit serum or anti-IPSE/alpha-1 mouse monoclonal antibody. The negative control (neg. control) was incubated without primary antibody/serum, but with secondary antibody. Membranes were imaged using chemiluminescence and a Fujifilm LAS-4000.</p

In gel detection of five different <i>S. mansoni</i> antigens expressed in vitro using WGL.

<p>SPO-1: Smp_113760; GST-26k: Smp_102070; TSP-1, extracellular loop 2: Smp_095630; IPSE/alpha-1: Smp_112110; SmTAL1: Smp_045200; SmTAL2: Smp_086480. Details of sequences and expected molecular weights are given in the Supplementary Data in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003124#pntd.0003124.s007" target="_blank">Table S3</a>. Success of translation was monitored by incorporation of BODIPY-labelled fluorescent Lysine in separate aliquots during translation. Samples were run on 4–20% SDS-PAGE gradient gels under reducing conditions and imaged in a Fujifilm LAS-4000. The left lanes show the wheat germ lysate control without template DNA, either without (−K) or with Lysine incorporation (+K), indicating fluorescent components produced during <i>in vitro</i> translation from endogenous mRNA.</p

Activation of RS-ATL8 reporter system by IgE binding factor IPSE/alpha-1 expressed in wheat germ (white bars) and <i>E. coli</i> (black bars).

<p>All cells (except IgE only) were sensitized for 16 hours with pooled serum from <i>P. judaica</i> allergic individuals diluted 1∶50 (v/v). The experiment included positive controls: sensitized with 1 µg/mL IgE and stimulated with the indicated amounts of polyclonal anti-human IgE (hatched bars); stimulated with Par j 2 (100 pg/ml; grey bar), as well as the negative controls (grey bas): serum sensitized/unstimulated cells (serum only). Data are mean ±SD of the readings of three separate wells. Multiplicity adjusted p-values were obtained by ANOVA followed by Dunnett's test for each condition compared with serum only control ****: <i>p</i><0.0001; ***: p<0.001; n.s.: not significant.</p

Luminescence data from the determination of serum-mediated and detergent-mediated cytotoxicity.

<p>The Par j 2 serum pool was incubated with a mixture of 0.3% (v/v) TNBP (Tri-N-butylphosphate) and 1% (v/v) Tween-80 (Polyoxyethyleneorbitan) and incubated under rotation at room temperature for 1 hr. Treated and untreated serum samples were then used in the indicated dilutions (v/v) to sensitise RS ATL8 cells overnight, followed by stimulation with 100 pg/mL Par j 2 recombinant allergen the next day. Data are mean ±SD of the readings of three separate wells. Positive and negative controls included in this experiment (A23187 and PMA: 1840.00±168.02 RLU; IgE+anti-IgE: 4991.00±171.62 RLU) are not shown. N.s.: not significant; n.t.: not tested; *: p<0.05; ****: <i>p</i><0.0001 (ANOVA followed by Tukey post-hoc test, all p-values adjusted for multiplicity).</p

Luminescence data of RS-ATL8 assay demonstrating the allergenicity of SmTAL-1.

<p>Recombinant SmTAL1 and SmTAL2 were expressed in <i>E. coli</i> (rTAL1, rTAL2; both 10 ng/ml) and in WGL (WG TAL1, WG TAL2; diluted 1∶10⁴). Cells were sensitized for 16 hours with pooled, virally inactivated plasma from individuals living in a <i>S. mansoni</i> endemic area in Uganda (diluted 1∶100) and stimulated with the recombinant antigens. The experiment included the negative controls: unsensitized and unstimulated cells (medium), cells incubated only with treated plasma diluted 1∶100 v/v (serum) and the positive control (IgE+aIgE, both 1 µg/mL). Data are mean +SD of the readings of three separate wells. Multiplicity adjusted p-values were obtained by ANOVA followed by Dunnett's test for each condition compared with plasma only control. ****: <i>p</i><0.0001; ***: p<0.001; n.s.: not significant.</p

Optimal concentration and minimal concentration of detectable allergen.

<p>This was determined by sensitising RS-ATL8 cells with the pooled serum (1∶50 dilution) from individuals with a specific IgE response to the Par j 2 allergen and challenged with recombinant Par j 2, serially diluted from 1 ng/mL to 10 fg/mL in 1∶10 dilution increments. Data are mean ±SD of the readings of three separate wells. Multiplicity adjusted p-values (ANOVA followed by Dunnett's post-hoc test) ***: <i>p</i><0.001, *: <i>p</i><0.05, n.s.: not significant. Representative of three separate experiments performed in triplicates with comparable results.</p

Correlation between the IgE titre measured in each plasma sample and the intensity of luciferase induction after stimulation with SmTAL-1.

<p>The Spearman rank correlation coefficient was R_s = 0.663 (p = 0.035, two-sided).</p