Analysis of the IgG and IgE reactivity of Fra e 9 and Ole e 9.

<p>(A) Identification of the isoforms pattern of both β-1,3-glucanases by 2-DE with pAbs raised against rNtD and rCtD of Fra e 9 in ash and olive pollen extracts. (B) IgE-binding inhibition analysis by ELISA of Fra e 9 recombinant domains after preincubation of the pool of sera from patients allergic to olive with increasing amounts of ash or olive pollen extracts.</p

Alignment of Fra e 9 with the β-1,3-glucanases from other plant sources.

<p>Ash (Fra e 9, KC920916), olive (Ole e 9, Q94G86), pear (B9VQ36), <i>A</i>. <i>thaliana</i> (Q06915), latex (Hev b 2, A2TJX4), banana (Mus a 5, A7U7Q7), tomato (Q01413), barley (P15737). (A) Dashes indicate gaps. Letters over black shading are conserved residues in all sequences; dark gray indicates residues conserved in at least six sequences; light gray indicates residues conserved in five sequences. % I and %S represent identity and similarity percentages of these sequences comparing to that of Fra e 9. (B) Independent comparison of the amino acid sequences of N-terminal and C-terminal domains of the β-1,3-glucanases with Fra e 9 domains.</p

Recombinant expression and molecular characterization of CtD-Fra e 9.

<p>(A) Time-course of the expression of rCtD-Fra e 9 in <i>P</i>. <i>pastoris</i>. Extracellular medium was harvested at different times after induction and stained with Coomassie Blue (CBS) after SDS-PAGE. (B) Purified domain was analysed by CBS, immunostaining with a pool of sera from olive pollen allergic patients (Sera) and with a Ole e 9-specific antiserum (pAb), and staining with ConA-rNtD was loaded in the same lane of the gel as a control for no glycosylation-. (C) Mass spectrometry analysis. (D) CD spectra in the far-UV (190–250 nm). (E) thermal unfolding assay during heating from 20°C to 80°C as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133066#pone.0133066.g004" target="_blank">Fig 4</a>. (F) Schematic representation of the 3D of Fra e 9 modeled against Ole e 9 3D structure determined by NMR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133066#pone.0133066.ref026" target="_blank">26</a>]. (G) Affinity gel electrophoresis (AGE) analysis of rCtD-Fra e 9 (Fra e 9), rCtD-Ole e 9 (Ole e 9) and BSA as negative control; both proteins were electrophoresed under non-denaturing conditions in polyacrylamide gels in the presence and absence (-) of laminarin.</p

Nucleotide sequence of cDNA encoding Fra e 9 and deduced amino acid sequence.

<p>The putative cleavage site for the signal peptide is indicated by an arrowhead. The catalytic residues are boxed. The cysteine residues of the C-terminal domain are circled. The potential N-glycosylation sites are framed.</p

Recombinant expression and molecular and functional characterization of the NtD-Fra e 9.

<p>(A) Time-course of the expression of rNtD-Fra e 9 in <i>E</i>. <i>coli</i>. Supernatants and pellets from cultures were harvested at different times after induction and stained with Coomassie Blue (CBS) after SDS-PAGE. (B) Purified domain was analysed by CBS and by immunostaining with a pool of sera from olive pollen allergic patients (Sera) or specific antiserum (pAb). (C) Mass spectrometry analysis. (D) CD spectrum in the far-UV (190–250 nm). In the inset are included the porcentages of secondary structure. (E) Thermal unfolding measured as ellipticity at 220nm during heating from 20°C to 80°C. (F) Schematic representation of the 3D-model of NtD-Fra e 9 and NtD-Ole e 9; The main structural differences are highlighted. (G) The pH dependence for the enzymatic activity of rNtD-Fra e 9 was assayed at different pH values.</p

Detection of a β-1,3-glucanase homolog to Ole e 9 in ash pollen.

<p>Coomassie Blue staining (CBS), IgG reactivity of pAbs obtained agaisnt rNtD and rCtD of Ole e 9 and IgE reactivity of a pool of sera from patients allergic to olive pollen, to olive and ash pollen extracts (40 μg of total protein). Molecular mass markers are indicated.</p

Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H₂O₂ was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a sensitivity higher than that of a commercial standard ELISA using a just-in-time produced protein in a simpler protocol with less sample volume and easily miniaturized and cost-effective instrumentation

Co-sensitization graph of TLP allergens.

<p>Each node represents one allergen (TLP as white ellipses, non-TLP allergens as blue square nodes, and LTP-allergen Pru p 3 as a green diamond) and the links represent co-sensitization of one or more sera for the linked allergens. The weight of each link, ranging between 0 and 1, measures the degree of co-sensitization. For the sake of clarity, only the 25 links of weight greater than 0.50 out of the total 253 existing links were plotted.</p

Average weight of allergens included in the co-sensitization graph.

<p>Average weight of allergens included in the co-sensitization graph.</p

Pollen counts (grains/m³ of air) of the regions included in the study.

<p>Data were obtained as the average of the previous n years, from the Comité de Aerobiología-SEAIC (<a href="http://www.polenes.com/concentraciones.html" target="_blank">http://www.polenes.com/concentraciones.html</a>) and from PIA-Punto de información de Aerobiología-UAB (<a href="http://lap.uab.cat/aerobiologia/" target="_blank">http://lap.uab.cat/aerobiologia/</a>).</p>*<p>Average of pollen counts (grains/m³). The time period is indicated in brackets.</p