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

IgE reactivity and allergenic activity of rDer p 18.

<p>(A) IgE levels (y-axis: ISU ISAC standardized units) specific for a panel of HDM allergens (x-axis: Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 11, Der p 14, Der p 15, Der p 18, Der p 21 and Der p 23) in HDM-allergic patients. (B) Dot-blotted rDer p 2, rDer p 18 and BSA were tested for IgE reactivity with sera from 7 HDM-allergic patients positive to rDer p 18 (#92–98), serum of a non-allergic person (NC) and with buffer (BC). Bound IgE Abs were detected with ¹²⁵I-labeled anti-human IgE Abs and visualized by autoradiography. (C) Up-regulation of CD203c expression was determined by FACS analysis after incubation of basophils from 4 HDM-allergic patients (#92–95) with increasing concentrations of rDer p 2 or rDer p 18 (x-axes) and displayed as stimulation index on the y-axes. (D) Recombinant Der p 18, rDer p 2, rDer p 23 and BSA were dotted onto nitrocellulose strips and incubated with sera from Der p 18-positive patients (#99, #100). Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human antibodies and visualized by autoradiography. (E) Inhibition of IgE reactivity to blotted nDer p 18 by rDer p 18. Nitrocellulose-blotted <i>D</i>. <i>pteronyssinus</i> extract was incubated with sera from Der p 18-sensitized patients (#99 and #100), which had been pre-incubated with rDer p 18, or for control purposes, with BSA. Molecular weights (kDa) were shown at the margins.</p

Percentages of amino acid sequence identities among chitinases and chitinase-like proteins from mites, insects, crustaceans and human¹.

<p>Percentages of amino acid sequence identities among chitinases and chitinase-like proteins from mites, insects, crustaceans and human<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160641#t001fn001" target="_blank">¹</a>.</p

Characterization of purified rDer p 18.

<p>(A) An aliquot of 3 μg of rDer p 18 was separated by SDS-PAGE under reducing (1) and non-reducing (2) conditions and stained with Coomassie brilliant blue. M, molecular weight marker. (B) Far UV CD analysis of rDer p 18. The graph represents the mean residue ellipticity (θ, y-axis) at a given wavelength (190–250 nm, x-axis).</p

Structural alignment.

<p>(A) Alignment of predicted secondary structure elements of Der p 18 with the secondary structure of human chitinase (1wawA) for portions of human chitinase with known three-dimensional structure and of the Der p 18 putative chitin-binding domain with tachycitin (1dqcA) as created with the SWISS-MODEL program. Identical amino acids are indicated by dots, dashes represent gaps and similar secondary structure elements (β-strands, α-helices) are marked. (B) Structural model of Der p 18 generated by the SWISS-MODEL program. The 29-amino-acid connecting sequence is indicated by a continuous line.</p

Chitin-binding activity of Der p 18.

<p>Coomassie-stained SDS-PAGE containing aliquots of rDer p 18, rDer p 15, WGA (positive control) and rDer p 5 (negative control) or HDM extract (lanes 1), the supernatants of these proteins (lanes 2) and proteins eluted from the chitin/chitin beads (lanes 3). The molecular weight marker is shown in lanes M. (C) Nitrocellulose-blotted samples of the experiment performed with HDM-extract and chitin beads (B) were incubated with rabbit anti-Der p 18 or anti-Der p 15 antibodies.</p