Allergens in allergy diagnosis: a glimpse at emerging new concepts and methodologies

Allergic diseases are important concern of public health. A reliable diagnosis is of utmost importance for the management of allergic patients both when immunotherapy is planned and when the treatment is essentially based on the avoidance of the allergy source. However, the available diagnostic systems sometimes fail to detect specific IgE antibodies thus impairing the correct diagnosis. The traditional test systems are generally based on the use of protein extracts derived from the allergenic sources whose composition is very variable and cannot be standardized. The development of a new methodology combining the so-called allergenic molecule-based diagnosis with the multiplex microarray technology and allowing the analysis of multiple purified allergens in a single test represents an important improvement in allergy diagnosis. In addition, the biochemical and immunological characterisation of individual allergens has provided new insights into the understanding of allergen-IgE recognition that could be exploited for further improvements of allergy diagnostic tests

Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

Background: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. Objective and Methods: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Moleculebased micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. Results: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE t

IgE Recognition Patterns of Profilin, PR-10, and Tropomyosin Panallergens Tested in 3,113 Allergic Patients by Allergen Microarray-Based Technology

BACKGROUND: IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed. METHODS: We evaluated the IgE recognition profile of profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2), PR-10 proteins (Aln g 1, Api g 1, Bet v 1.0101, Bet v 1.0401, Cor a 1, Dau c 1 and Mal d 1.0108) and tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7) using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion. RESULTS: 3,113 individuals (49.9% female) were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgE(+)), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE(+)) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE(+)). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis. CONCLUSIONS: Unless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients' immunological phenotypes

Age group distribution of the ISAC IgE panallergens-reactive population; n = 3,113.

<p>Black bars: Total 23,077 original allergic population; White Bars: Total 3,113 panallergens allergic population; Dark grey bars: PR-10; Hatched bars: Profilin; Light grey bars: Tropomyosin.</p

ABA <i>versus</i> ISAC correlation results on 137 serum samples selected on the basis of nDer s 1, nPen m 1, and nPru p 3 mutually exclusive IgE positivity are reported.

<p>Panel A: All 411 IgE values, obtained by testing the three allergens; Panel B: 137 IgE results obtained on nDer s 1 allergen; Panel C: 137 IgE results obtained on nPen m 1 allergen; Panel D: 137 IgE results obtained on nPru p 3 allergen. For graphical visualization needs on log scales, zero values for ABA were set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC values on the Y axis. The Spearman r correlation coefficient, the χ² and the Fisher's exact tests were used where applicable.</p

Bivariate analysis of reciprocal relationships of IgE to profilin, PR-10, and tropomyosin panallergens.

<p>The number of IgE reactive subjects and the Pearson coefficient are shown for paired allergens within the three panallergen groups. Pearson r values are reported below. Data were statistically significant in all cases (p<0.001). Panel A: Profilins; Panel B: PR-10; Panel C: Tropomyosins.</p

ABA <i>versus</i> ISAC correlation results on serum samples selected on the basis of the allergen specificities reported in each panel and listed in Table S1.

<p>Letter flags, namely N, O, P, Q, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035697#pone-0035697-g008" target="_blank">figure 8</a> indicate them as parts of the results shown also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035697#pone-0035697-g005" target="_blank">figures 5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035697#pone-0035697-g006" target="_blank">6</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035697#pone-0035697-g007" target="_blank">7</a>. Consecutive letters are used on purpose to recall result type continuity across the four figures. Allergen nature, being either natural or recombinant, matched for both tests. Vertical dashed lines represent the arbitrary ABA negative cut off value. Horizontal dashed lines mark the value range where ISAC IgE determinations are not always reproducible (unpublished data). For graphical visualization needs on log scales, zero value for ABA was set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC value on the Y axis. The Spearman r correlation coefficient was calculated and the Fisher's exact test was used for statistical purposes. Statistical results are reported below each graph.</p