T cell responses to bGST and Bet v 1 in BALB/c mice.

<p>(A) Proliferative and cytokine responses of splenocytes from mice immunized with bGST (n = 5) to titrated amounts of bGST, BPE (25 µg/mL) and Bet v 1 (5 µg/mL). Cytokines were induced with 1.25 µg/mL of bGST. (B) Proliferative and cytokine responses of splenocytes from mice immunized with Bet v 1 (n = 5) to Bet v 1, BPE (25 µg/mL) and bGST (1.25 µg/mL). Cytokines were induced with 5 µg/mL of Bet v 1. Δcpm, cpm of unstimulated cultures were subtracted from stimulated cultures (mean background cpm = 4724±742); Δpg/mL, levels in unstimulated cultures were subtracted from stimulated cultures (mean pg/mL of IFN-γ were <135 pg/ml and of remaining cytokines <20 pg/ml).</p

No cross-reactivity between bGST and HDM-GST.

<p>(A) IgG1-reactivity of sera (n = 3) collected from bGST-immunized mice at day 0 and day 59 to BPE and HDME. (B) IgE-reactivity of seven Der p 8-sensitized HDM-allergic patients to bGST and HDME. NHS, non-allergic control sera; O.D. optical density; dotted and dashed lines indicate the cut-off for positive IgE-reactivity.</p

Ab responses to bGST and Bet v 1 in BALB/c mice.

<p>(A) bGST-specific Ig of mice immunized with bGST (n = 5). (B) Bet v 1-specific Ig of mice immunized with Bet v 1 (n = 5). (C) IgG1 responses to BPE from mice immunized with either bGST or Bet v 1. O.D.; optical density, d, day; mean values+standard deviation are shown.</p

Purity and identity of bGST.

<p>(A) Coomassie-stained SDS-PAGE showing bGST before (lanes 1 and 2) and after Ni²⁺ affinity-chromatography (lane 3). (B) Circular dichroism spectroscopy of bGST. Far UV-measurements of bGST were recorded at 20°C (solid line), 95°C (dashed line) and 20°C after renaturation (dotted line). The mean residue ellipticities (⊖) are shown at given wavelengths. (C) Natural bGST was excised from a Coomassie-stained SDS-PAGE of BPE and analysed by mass spectrometry. Bold characters show aa residues of natural bGST in the sequence of recombinant bGST.</p

IgE-responses of BP-allergic individuals.

<p>(A) IgE-reactivity of 217 BP-allergic patients to BP allergens and bGST. (B) IgE-reactivity to bGST is inhibited by pre-incubation of sera with BPE (gray line) and bGST (black line). One representative example of four is shown. (C) RBL assay with sera from three patients sensitized to bGST and Bet v 1. Degranulation was induced with titrated amounts of bGST (filled circles) and Bet v 1 (open circles). The release of β-hexosaminidase is expressed as percentage of the release from anti-IgE-treated cells.</p

Inhibition of allergic patients' IgE binding to Tri a 37 by rabbit anti-Tri a 37 antibodies.

<p>Tri a 37 was tested for IgE reactivity with sera from three Tri a 37-allergic patients (1, 3, 4), serum from a non-allergic individual or buffer after pre-incubation with rabbit anti-Tri a 37 antibodies (Immune) or the rabbits pre-immune serum (Pre). Shown are the optical density (O.D.) levels corresponding to bound IgE.</p

Antibody-recognition of EcTri a 37, BvTri a 37 and Tri a 37 peptides.

<p>(<b>A</b>) Nitrocellulose-dotted wheat seed extract (WSE), human serum albumin (HSA), EcTri a 37 and BvTri a 37 were tested with sera from ten wheat food allergic patients (1–10), with serum from a non-allergic individual (N), buffer alone (B), with Tri a 37-specific rabbit antibodies (I), the corresponding rabbit pre-immune serum (P) and buffer control (B). (<b>B</b>) Testing of Tri a 37 peptides (P1–P5) as in (A).</p

Demographic, clinical and serological characterization of wheat food-allergic patients.

<p>Demographic, clinical and serological characterization of wheat food-allergic patients.</p

Cross-reactivity of rabbit anti-rDer p 18 IgG antibodies with proteins from other mites, crustacea, mollusca and insects.

<p>Blots containing extracts from <i>Dermatophagoides pteronyssinus</i>, <i>Dermatophagoides farinae</i>, <i>Blomia tropicalis</i>, shrimp, lobster, snail and wasp which had been separated by SDS-PAGE were incubated with normal rabbit antibodies before immunization (A), with rabbit anti-rDer p 18 (B) or with rabbit anti-rDer p 10 antibodies (C). (D) Inhibition of IgG reactivity to blotted nDer p 18 and nDer f 18. Nitrocellulose-blotted <i>D</i>. <i>pteronyssinus</i> (left panel) and <i>D</i>. <i>farinae</i> (right panel) extracts were incubated with a rabbit anti-Der p 18 pre-immune serum or immune serum, which had been pre-incubated with <i>D</i>. <i>pteronyssinus</i> extract, <i>D</i>. <i>farinae</i> extract, rDer p 18, rDer p 15 or BSA. Molecular weights (kDa) are shown at the margins.</p

Characterization of purified Tri a 37 expressed in <i>E.coli</i> cells (EcTri a 37) and in baculovirus-infected insect cells (BvTri a 37) as well as detection of natural Tri a 37 in wheat seed extract.

<p>(<b>A</b>) Coomassie brilliant blue-stained SDS-PAGE under reducing (lane R) and under non-reducing (lane NR) conditions. A protein molecular weight marker (kDa) is shown on the left side. (<b>B</b>) Detection of IgE-reactivitiy to Western-blotted EcTri a 37 and BvTri a 37, using serum from a wheat food allergic patient (P) and buffer (B) as a control. Western-blotted wheat seed extract was tested with Tri a 37-specific rabbit antibodies (Immune) or the corresponding pre-immune serum (Pre). Bound human IgE and rabbit IgG antibodies were detected with ¹²⁵Iodine-labeled antibodies and visualized by autoradiography. Molecular weights are indicated in kilo Dalton (kDa). (<b>C</b>) Mass spectrometry of recombinant EcTri a 37 and BvTri a 37. The mass/charge ratios are shown on the <i>x</i>-axes, and the intensities are displayed on the <i>y</i>-axes as percentages of the most intensive signals obtained in the investigated mass range. (<b>D</b>) Circular dichroism analysis of recombinant EcTri a 37 and BvTri a 37. The spectra are expressed as mean residue ellipticities (θ) (<i>y</i>-axes) at given wavelengths (<i>x</i>-axes).</p