RESUMEN

Background: Asthma is not well investigated in equatorial Africa and little is known about the disease-associated allergen molecules recognized by IgE from patients in this area. The aim was to study the molecular IgE sensitization profile of asthmatic children and young adults in a semi-rural area (Lambaréné) of an equatorial African country (Gabon), to identify the most important allergen molecules associated with allergic asthma in equatorial Africa. Methods: Fifty-nine asthmatic patients, mainly children and few young adults, were studied by skin prick testing to Dermatophagoides pteronyssinus (Der p), D. farinae (Der f), cat, dog, cockroach, grass, Alternaria and peanut. Sera were obtained from a subset of 35 patients, 32 with positive and 3 with negative skin reaction to Der p and tested for IgE reactivity to 176 allergen molecules from different allergen sources by ImmunoCAP ISAC microarray technology and to seven recombinant Blomia tropicalis (Blo t) allergens by IgE dot blot assay. Results: Thirty-three of the 59 patients (56%) were sensitized to Der p and 23 of them (39%) were also sensitized to other allergen sources, whereas 9 patients (15%) were only sensitized to allergen sources other than Der p. IgE serology analyses (n=35) showed high IgE-binding frequencies to the Blo t allergens Blo t 5 (43%), Blo t 21 (43%) and Blo t 2 (40%), whereas the Der p allergens rDer p 2, rDer p 21 and rDer p 5 (34%, 29% and 26%) were less frequently recognized. Only few patients showed IgE reactivity to allergens from other allergen sources, except to allergens containing carbohydrate determinants (CCDs) or to wasp venom allergens (i.e., antigen 5). Conclusion: Our results thus demonstrate that IgE sensitization to mite allergens is very prevalent in asthmatics in Equatorial Africa with B. tropicalis allergen molecules representing the most important ones associated with allergic asthma.

Asunto(s)

Alérgenos , Asma , Animales , Perros , Inmunoglobulina E , Dermatophagoides farinae , Gabón

RESUMEN

BACKGROUND: Immunoglobulin-E(IgE)-mediated hypersensitivity to cow's milk allergens is a frequent cause of severe and life-threatening anaphylactic reactions. Besides case histories and controlled food challenges, the detection of the IgE antibodies specific to cow's milk allergens is important for the diagnosis of cow-milk-specific IgE sensitization. Cow´s milk allergen molecules provide useful information for the refined detection of cow-milk-specific IgE sensitization. METHODS: A micro-array based on ImmunoCAP ISAC technology was developed and designated milk allergen micro-array (MAMA), containing a complete panel of purified natural and recombinant cow's milk allergens (caseins, α-lactalbumin, ß-lactoglobulin, bovine serum albumin-BSA and lactoferrin), recombinant BSA fragments, and α-casein-, α-lactalbumin- and ß-lactoglobulin-derived synthetic peptides. Sera from 80 children with confirmed symptoms related to cow's milk intake (without anaphylaxis: n = 39; anaphylaxis with a Sampson grade of 1-3: n = 21; and anaphylaxis with a Sampson grade of 4-5: n = 20) were studied. The alterations in the specific IgE levels were analyzed in a subgroup of eleven patients, i.e., five who did not and six who did acquire natural tolerance. RESULTS: The use of MAMA allowed a component-resolved diagnosis of IgE sensitization in each of the children suffering from cow's-milk-related anaphylaxis according to Sampson grades 1-5 requiring only 20-30 microliters of serum. IgE sensitization to caseins and casein-derived peptides was found in each of the children with Sampson grades of 4-5. Among the grade 1-3 patients, nine patients showed negative reactivity to caseins but showed IgE reactivity to alpha-lactalbumin (n = 7) or beta-lactoglobulin (n = 2). For certain children, an IgE sensitization to cryptic peptide epitopes without detectable allergen-specific IgE was found. Twenty-four children with cow-milk-specific anaphylaxis showed additional IgE sensitizations to BSA, but they were all sensitized to either caseins, alpha-lactalbumin, or beta-lactoglobulin. A total of 17 of the 39 children without anaphylaxis lacked specific IgE reactivity to any of the tested components. The children developing tolerance showed a reduction in allergen and/or peptide-specific IgE levels, whereas those remaining sensitive did not. CONCLUSIONS: The use of MAMA allows for the detection, using only a few microliters of serum, of IgE sensitization to multiple cow's milk allergens and allergen-derived peptides in cow-milk-allergic children with cow-milk-related anaphylaxis.

Asunto(s)

Anafilaxia , Hipersensibilidad a la Leche , Animales , Femenino , Bovinos , Leche , Alérgenos , Caseínas , Lactalbúmina , Anafilaxia/diagnóstico , Inmunoglobulina E , Péptidos , Lactoglobulinas , Proteínas de la Leche

RESUMEN

BACKGROUND: Molecular antibody reactivity profiles have not yet been studied in depth in patients treated by sublingual house dust mite (HDM) tablet immunotherapy. Humoral immune responses to a large panel of HDM mite allergens were studied using allergen microarray technology in a subset of clinically defined high and low responder patients from a double-blind placebo-controlled allergen-specific immunotherapy (AIT) trial using sublingual 300 IR HDM tablets. METHODS: Serum levels of IgE, IgG and IgG4 to 13 Dermatophagoides pteronyssinus molecules were measured at baseline and after 1-year AIT, using allergen microarrays in 100 subjects exhibiting high or low clinical benefit. RESULTS: Der p 1, Der p 2 and Der p 23 were the most frequently recognized allergens in the study population. Patients with HDM-related asthma had significantly higher allergen-specific IgE levels to Der p 1 and Der p 23. No significant difference in the distribution of allergen sensitization pattern was observed between high and low responders. An increase in serum allergen-specific IgG and IgG4 occurred upon AIT, in particular to allergens Der p 1, Der p 2 and Der p 23 (p < 0.0001). CONCLUSIONS: We confirm for our study population that Der p 1- and Der p 23-specific IgE levels are associated with asthma. IgE reactivity profiles were not predicitive of sublingual AIT outcomes, with 300 IR tablets as efficacious in pauci- and multi-sensitized subjects. Our study is the first to demonstrate the induction of IgG and IgG4  specific for the HDM allergens Der p 1, Der p 2 and Der p 23 by sublingual AIT.

Asunto(s)

Asma , Inmunoterapia Sublingual , Alérgenos , Animales , Antígenos Dermatofagoides , Asma/terapia , Humanos , Inmunoglobulina E , Inmunoglobulina G , Factores Inmunológicos , Piridinolcarbamato , Pyroglyphidae , Comprimidos

RESUMEN

BACKGROUND: Previous studies demonstrated that birch pollen-related foods can cause late eczematous responses in birch pollen-sensitized patients with atopic dermatitis (AD). However, suitable markers to predict birch pollen-related food allergy in patients with AD are still lacking. OBJECTIVE: We evaluated the correlation of the results from ImmunoCAP® fluorescence enzyme immunoassay (FEIA) singleplex and ImmunoCAP® immuno solid-phase allergen chip (ISAC) multiplex system in AD patients and investigated the diagnostic validity of allergen microarray analysis, measuring specific IgE (sIgE) with ImmunoCAP® ISAC to predict birch pollen-related food allergy in patients with AD. METHODS: A total of 19 children and adults with AD, existing IgE-mediated birch pollen sensitization, and suspected birch pollen-related food allergy underwent a double-blind placebo-controlled food challenge (DBPCFC) in the clinical routine. Total and sIgE levels to birch pollen, Bet v 1, Bet v 2, and birch pollen-related foods (apple, carrot, celery, and hazelnut) were determined prior to the DBPCFC by ImmunoCAP®-FEIA. Additionally, allergen microarray ImmunoCAP® ISAC analysis was performed. Data were analyzed retrospectively. RESULTS: Twelve out of 19 patients (63% responders) experienced an allergic reaction upon DBPCFC. Overall, 7 patients (37%) developed a significant deterioration of AD with a median increase of 12.4 points in the scoring of atopic dermatitis (SCORAD) index (range 10.0-15.7). Oral allergy syndrome was the predominant immediate-type symptom (n = 11/12 responders). There were no differences in sensitization frequencies regarding allergens of the pathogenesis-related protein family 10 between responders and non-responders. In all patients, correlation of IgE levels determined with ImmunoCAP® ISAC and ImmunoCAP®-FEIA, respectively, was significant with high correlation coefficients regarding birch pollen allergen extract, rBet v 1, and rBet v 2 (rs > 0.8, p < 0.001) and lower but also significant correlation coefficients regarding food allergens (rs < 0.8, p < 0.05-<0.001). CONCLUSION: ImmunoCAP® ISAC microarray allows displaying a differentiated sensitization profile in birch pollen-sensitized patients with AD. However, IgE-mediated sensitization against birch pollen-related allergens revealed by the allergen multiplex system does not predict late eczematous reactions upon DBPCFC with birch pollen-related foods.

Asunto(s)

Dermatitis Atópica , Hipersensibilidad a los Alimentos , Adulto , Alérgenos , Betula , Niño , Dermatitis Atópica/diagnóstico , Hipersensibilidad a los Alimentos/diagnóstico , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina E , Análisis por Micromatrices , Polen , Estudios Retrospectivos

RESUMEN

Several factors can affect the allergen content and profile of a specific food, including processing procedures often leading to a decrease in allergenicity, although no change, or even an increase, have also been reported. Evaluation of the effectiveness of a processing procedure requires the availability of reliable methodologies to assess the variation in molecules able to induce allergic reactions in the analyzed food. Conventional and innovative strategies and methodologies can be exploited to identify allergenic proteins in foodstuffs. However, depending on the specific purposes, different methods can be used. In this review, we have critically reviewed the advantages of an innovative method, the multiplex allergen microarray-based immunoassay, in the detection of allergens in foodstuffs. In particular, we have analyzed some studies reporting the exploitation of an IgE-binding inhibition assay on multiplex allergen biochips, which has not yet been reviewed in the available literature. Unlike the others, this methodology enables the identification of many allergenic proteins, some of which are still unknown, which are recognized by IgE from allergic patients, with a single test. The examined literature suggests that the inhibition test associated with the multiplex allergen immunoassay is a promising methodology exploitable for the detection of IgE-binding proteins in food samples.

RESUMEN

BACKGROUND: Sensitization to house dust mite (HDM) is a leading cause of allergic rhinitis and asthma. Despite more than 30 HDM-derived allergens having been identified to date, specific therapeutic approaches do not yet take into account the local sensitization profiles of patients. This study aimed to identify patterns of HDM sensitization in HDM-allergic adults living in distinct geographic areas, to inform the development of targeted diagnostic and therapeutic tools. METHODS: Serum samples from 685 HDM-allergic subjects from Canada, Europe, South Africa, and the USA were tested for levels of IgE specific for 17 micro-arrayed HDM allergens by ImmunoCAP Immuno Solid-phase Allergen Chip (ISAC) technology. RESULTS: The results confirmed significant geographical variability in sensitization patterns and levels of IgE. In all areas, the major sensitizers were the group 1 and group 2 allergens and Der p 23. Der p 23 was a frequent sensitizer: 64% of the subjects had IgE specific for Der p 23, and 2.3% were monosensitized to it. In South Africa, Der p 23 was the dominant HDM allergen (86% prevalence) and Der p 7 achieved major allergen status (56%). IgE sensitization to HDM was influenced by asthmatic status, levels of allergen exposure, age, race-ethnicity and smoking status, but not by BMI. CONCLUSION: Sensitization profiles to HDM allergens differ considerably among distinct geographic areas, with Der p 7 and Der p 23 being major sensitizers in South Africa. Such heterogeneity should be taken into account in the diagnosis and treatment of HDM-allergic patients.

Asunto(s)

Inmunoglobulina E , Pyroglyphidae , Adulto , Alérgenos , Animales , Antígenos Dermatofagoides , Polvo , Europa (Continente) , Humanos , Sudáfrica/epidemiología

RESUMEN

BACKGROUND: House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. OBJECTIVE: Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. METHODS: Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. RESULTS: Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. CONCLUSION: Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.

Asunto(s)

Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Cisteína Endopeptidasas/inmunología , Desensibilización Inmunológica/métodos , Hipersensibilidad/terapia , Inmunoglobulina E/metabolismo , Inmunoglobulina G/metabolismo , Extractos Vegetales/uso terapéutico , Adulto , Animales , Epítopos/inmunología , Femenino , Humanos , Hipersensibilidad/inmunología , Inyecciones Subcutáneas , Masculino , Análisis por Matrices de Proteínas , Pyroglyphidae

RESUMEN

Previous reports suggested that ex vivo cultured primary nasal epithelial cells from allergic patients differ from those from non-allergic individuals by genuinely reduced barrier function. By contrast, we found that primary nasal epithelial cells from allergic and non-allergic individuals showed comparable barrier function and secretion of cytokines.

RESUMEN

Previous reports suggested that ex vivo cultured primary nasal epithelial cells from allergic patients differ from those from non-allergic individuals by genuinely reduced barrier function. By contrast, we found that primary nasal epithelial cells from allergic and non-allergic individuals showed comparable barrier function and secretion of cytokines.

Asunto(s)

Humanos , Citocinas , Células Epiteliales , Inmunoglobulina E , Rinitis Alérgica

RESUMEN

INTRODUCTION: Allergen-specific immunoglobulin isotype formation associated with immunoglobulin class-switching during the lactation period is the immunological background for food allergy in infants. We analyzed the serial changes in the production of feeding type-related egg- and milk-specific immunoglobulin isotypes from birth to 6 months of age with or without eczema in 84 infants. METHODS: Allergen-specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, IgA, and IgE levels of hen's egg and bovine milk were measured in cord blood and blood samples from infants at 2, 4, and 6 months of age by the densely carboxylated protein microarray. RESULTS: Formula and mixed feeding were associated with a rapid increase in cow's milk allergen-specific immunoglobulins and feeding type-related significant differences in casein-specific immunoglobulin levels were detected. Breast and mixed feeding were associated with slow but significant increase in ovalbumin-specific IgG1 and IgE levels, but not other immunoglobulins. We found two different immunoglobulin isotype formation at 6 months of age with low- or high-affinity IgE against ovalbumin. One isotype formation pattern had relatively high ovalbumin-specific IgG1 levels, detectable IgG2, and low-affinity IgE, while the other had low ovalbumin-specific IgG1 levels, undetectable IgG2, and high levels of high-affinity IgE. The incidence of eczema was significantly higher in the latter pattern (84.6%), compared with the remaining infants (42.2%). CONCLUSIONS: Feeding practice-related allergen sensitization and immunoglobulin isotype formation were identified during the lactation period. The development of eczema during the lactation period could potentially modify the immunoglobulin isotype formation with high levels of high-affinity IgE.

Asunto(s)

Alérgenos/inmunología , Eccema/inmunología , Hipersensibilidad al Huevo/inmunología , Huevos/efectos adversos , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulina E/inmunología , Hipersensibilidad a la Leche/inmunología , Leche/efectos adversos , Factores de Edad , Animales , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Bovinos , Pollos , Eccema/complicaciones , Hipersensibilidad al Huevo/complicaciones , Hipersensibilidad al Huevo/genética , Femenino , Humanos , Isotipos de Inmunoglobulinas/genética , Isotipos de Inmunoglobulinas/inmunología , Lactante , Recién Nacido , Masculino , Hipersensibilidad a la Leche/complicaciones , Hipersensibilidad a la Leche/genética , Embarazo

RESUMEN

BACKGROUND: In vitro detection of the allergen-specific IgE antibody (sIgE) is a useful tool for the diagnosis and treatment of allergies. Although multiple simultaneous allergen tests offer simple and low-cost screening methods, these platforms also have limitations with respect to multiplexibility and analytical performance. As an alternative assay platform, we developed and validated a microarray using allergen extracts that we termed "GOLD" chip. METHODS: Serum samples of 150 allergic rhinitis patients were used in the study, and the diagnostic performance of the microarray was compared with that of AdvanSure (LG Life Sciences, Daejun, Korea) and ImmunoCAP (Phadia, Uppsala, Sweden). Standard IgE samples were used for the quantitative measurement of sIgEs. RESULTS: The microarray-based assay showed excellent performance in the quantitative measurement of sIgEs, demonstrating a linear correlation within the range of sIgE concentrations tested. The limit of detection (LOD) was lower than 0.35 IU/mL, which is the current standard for the LOD cut-off. The assay also provided highly reproducible sets of data. The total agreement percentage of positive and negative calls was 92.2% compared with ImmunoCAP. Moreover, an outstanding correlation was observed between the microarray and the ImmunoCAP results, with Cohen's kappa and Pearson correlation coefficient values of 0.80 and 0.79, respectively. CONCLUSIONS: The microarray-based in vitro diagnostic platform offers a sensitive, reproducible, and highly quantitative method to detect sIgEs. The results showed strong correlations with that of ImmunoCAP. These results suggest that the new allergen microarray can serve as a useful alternative to current screening platforms, ultimately becoming a first-line screening method.

Asunto(s)

Alérgenos/inmunología , Inmunoglobulina E/sangre , Análisis por Micromatrices/métodos , Rinitis Alérgica/diagnóstico , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Inmunoensayo , Límite de Detección , Masculino , Persona de Mediana Edad , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Adulto Joven

RESUMEN

BACKGROUND: In vitro detection of the allergen-specific IgE antibody (sIgE) is a useful tool for the diagnosis and treatment of allergies. Although multiple simultaneous allergen tests offer simple and low-cost screening methods, these platforms also have limitations with respect to multiplexibility and analytical performance. As an alternative assay platform, we developed and validated a microarray using allergen extracts that we termed “GOLD” chip. METHODS: Serum samples of 150 allergic rhinitis patients were used in the study, and the diagnostic performance of the microarray was compared with that of AdvanSure (LG Life Sciences, Daejun, Korea) and ImmunoCAP (Phadia, Uppsala, Sweden). Standard IgE samples were used for the quantitative measurement of sIgEs. RESULTS: The microarray-based assay showed excellent performance in the quantitative measurement of sIgEs, demonstrating a linear correlation within the range of sIgE concentrations tested. The limit of detection (LOD) was lower than 0.35 IU/mL, which is the current standard for the LOD cut-off. The assay also provided highly reproducible sets of data. The total agreement percentage of positive and negative calls was 92.2% compared with ImmunoCAP. Moreover, an outstanding correlation was observed between the microarray and the ImmunoCAP results, with Cohen's kappa and Pearson correlation coefficient values of 0.80 and 0.79, respectively. CONCLUSIONS: The microarray-based in vitro diagnostic platform offers a sensitive, reproducible, and highly quantitative method to detect sIgEs. The results showed strong correlations with that of ImmunoCAP. These results suggest that the new allergen microarray can serve as a useful alternative to current screening platforms, ultimately becoming a first-line screening method.

Asunto(s)

Humanos , Disciplinas de las Ciencias Biológicas , Diagnóstico , Hipersensibilidad , Inmunoglobulina E , Técnicas In Vitro , Límite de Detección , Tamizaje Masivo , Métodos , Rinitis Alérgica

Asunto(s)

Aldehído-Liasas/inmunología , Artemia/inmunología , Peces/inmunología , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/inmunología , Inmunoglobulina E/inmunología , Hipersensibilidad Respiratoria/diagnóstico , Hipersensibilidad Respiratoria/inmunología , Animales , Artemia/enzimología , Electroforesis en Gel Bidimensional , Femenino , Hipersensibilidad a los Alimentos/terapia , Humanos , Inmunoglobulina E/sangre , Persona de Mediana Edad , Hipersensibilidad Respiratoria/terapia

RESUMEN

BACKGROUND: Allergen-specific IgE antibodies are a hallmark of type I allergy. The aim of this cross-sectional study was to analyze the sensitization profiles of an Austrian adolescent population utilizing molecule-based IgE diagnosis. METHODS: Serum samples of 501 nonselected pupils from Salzburg, Austria, were tested in ImmunoCAP ISAC® for IgE reactivity to 112 single allergens. Sensitization profiles were assessed and statistically coordinated with reported allergies. RESULTS: In the population aged 12-21 years, 53.5% showed IgE reactivity to at least one allergen tested. The highest prevalence was found for Phl p 1 from grass pollen (26.5%), group 2 mite allergens (18.2%), Bet v 1 from birch pollen (16.3%) and Fel d 1 from cat (14.4%). The majority of participants showed a complex sensitization profile and reacted on average to 9 allergens. Pollen sensitization was highly prevalent (41.7%) and mainly driven by group I grass and PR-10 allergens of the Betulaceae family, while Pla l 1 represented the most relevant weed. Diagnosed and self-reported allergies were noted in 21.9% and 45.5% of participants, respectively, and correlated well with in vitro results. Among atopic individuals, 71.4% reported to suffer from at least one allergy; concordance was found for grass and cat sensitization, while venom- and weed pollen-positive individuals were frequently asymptomatic. CONCLUSIONS: More than half of the tested adolescent population had already established an atopic status presenting a complex IgE reactivity profile dominated by pollen sensitization. Detailed molecule-based analysis allows determining relevant biomarkers and monitoring of the atopic status in populations.

Asunto(s)

Alérgenos/inmunología , Hipersensibilidad/epidemiología , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Adolescente , Adulto , Austria/epidemiología , Niño , Estudios Transversales , Femenino , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad Inmediata/epidemiología , Hipersensibilidad Inmediata/inmunología , Masculino , Prevalencia , Adulto Joven

RESUMEN

Allergen-specific immunotherapy (AIT) is the only treatment of IgE-mediated allergies so far that has a sustained effect on clinical symptoms and can modify the course of the disease. It is an allergen-specific treatment and therefore requires the correct identification of the disease-causing allergens. Furthermore, AIT is a time-consuming treatment for which the efficacy is dependent on several factors. Therefore, diagnostic tests and biomarkers are needed that facilitate (1) selection of the correct allergens according to the patient's individual sensitization profile and (2) to monitor the effects of AIT. This can provide support for the decision to continue, modify, or discontinue vaccination. One significant mechanism of action of AIT is the induction of allergen-specific antibodies that compete with IgE for the binding to allergen molecules, hence referred to as blocking antibodies. It was shown in several studies that the induction of blocking antibodies by AIT, and their specificity can be measured by allergen microarrays. Inhibition of allergen-specific IgE binding by blocking antibodies can also be determined by microarrays and is associated with changes in clinical parameters or other in vivo and in vitro assays demonstrating efficacy of AIT. Furthermore, allergen microarrays allow determination of IgE sensitizations towards a comprehensive set of allergen molecules and therefore are well suited for identifying the disease-causing allergens for correct prescription of AIT. Thus, diagnostic tests based on microarrayed allergens can be useful in determining the correct prescription of AIT and can be used to monitor efficacy of AIT.

RESUMEN

We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (~0.02 kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.

Asunto(s)

Autoanticuerpos/sangre , Glicoproteínas/sangre , Inmunoensayo/instrumentación , Inmunoglobulina E/sangre , Microquímica/instrumentación , Espectrometría de Fluorescencia/instrumentación , Animales , Autoanticuerpos/inmunología , Técnicas Biosensibles/instrumentación , Gatos , Diseño de Equipo , Análisis de Falla de Equipo , Glicoproteínas/inmunología , Humanos , Inmunoglobulina E/inmunología , Fotones

RESUMEN

BACKGROUND: House dust mites (HDMs) represent one of the most important inducers of respiratory allergies worldwide. OBJECTIVE: We sought to investigate the IgE and IgG reactivity profiles to a comprehensive panel of HDM allergens in children with allergic asthma and to compare them with those of nonasthmatic atopic children. METHODS: Sera from clinically well-characterized asthmatic children with HDM allergy (n = 105), nonasthmatic children (n = 53), and nonatopic nonasthmatic children (n = 53) were analyzed for IgE and IgG reactivity to a panel of 7 HDM allergens (nDer p 1, rDer p 2, rDer p 5, rDer p 7, rDer p 10, rDer p 21, and rDer p 23) by means of allergen microarray technology. RESULTS: Asthmatic children with HDM allergy more frequently showed an IgE response to each of the HDM allergens and recognized more allergens than nonasthmatic children with HDM allergy. Furthermore, IgE levels to certain HDM allergens (nDer p 1, P = .002; rDer p 2, P = .007; rDer p 5, P = .031; and rDer p 23, P < .001) were significantly higher in asthmatic children than in children without asthma. By contrast, fewer asthmatic children showed IgG reactivity to HDM allergens than nonasthmatic children, but allergen-specific IgG levels were comparable. CONCLUSION: The IgE and IgG reactivity profiles to HDM allergens, as well as IgE levels to certain allergen components, differed considerably between children with and without asthmatic symptoms caused by HDM allergy. In fact, asthmatic children were characterized by an expanded IgE repertoire regarding the numbers of recognized allergen components and by increased specific IgE levels.

Asunto(s)

Alérgenos/inmunología , Especificidad de Anticuerpos , Antígenos Dermatofagoides/inmunología , Asma/inmunología , Inmunoglobulina E/inmunología , Adolescente , Animales , Asma/sangre , Niño , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratas

RESUMEN

BACKGROUND: Peanut allergy (PA) management was improved by the introduction of molecular allergology, but guidelines for Mediterranean patients are lacking. We aimed at evaluating peanut component-resolved diagnosis as a diagnostic and prognostic tool in children from Southern France. METHODS: In 181 pediatric patients, PA diagnosis was founded on medical history, skin prick testing, serum-specific IgE to Arachis hypogea extract and components, Pru p 4, and plant carbohydrates, and oral food challenge. Allergen microarray was also performed in 68 of these patients. RESULTS: In peanut-allergic children (n = 117), IgE to Ara h 6 were most prevalent (64%), followed by Ara h 2 (63%), Ara h 1 (60%), and Ara h 9 (52%). Ara h 6 was the best predictor of PA. The second best predictor was the ratio of Ara h 2 IgE to peanut IgE (cutoff 0.113). Persistent childhood PA was associated with complex molecular profiles. Comparison of singleplex and microarray results showed poor concordance for Ara h 2 and Ara h 9. CONCLUSION: Ara h 6 and Ara h 2 are the best predictors of PA at diagnosis in Mediterranean pediatric patients. Ara h 1, Ara h 8, and molecular complexity are associated with PA persistence.

Asunto(s)

Albuminas 2S de Plantas/inmunología , Antígenos de Plantas/inmunología , Glicoproteínas/inmunología , Hipersensibilidad al Cacahuete/diagnóstico , Adolescente , Arachis , Proteínas Portadoras/inmunología , Niño , Preescolar , Femenino , Francia , Humanos , Inmunización , Inmunoglobulina E/sangre , Lactante , Recién Nacido , Masculino , Región Mediterránea , Análisis por Micromatrices , Proteínas de Plantas/inmunología , Guías de Práctica Clínica como Asunto , Valor Predictivo de las Pruebas , Pronóstico

RESUMEN

The knowledge on molecular allergy diagnosis is continuously evolving. It is now time for the clinician to integrate this knowledge and use it when needed to improve the accuracy of diagnosis and thus provide more precise therapeutic and avoidance measures. This review does not intend to comprehensively analyze all the available allergen molecules, but to provide some practical clues on use and interpretation of molecular allergy diagnosis. The potential role of component resolved diagnosis in circumstances such as the indication of allergen immunotherapy, pollen polysensitization, food allergy, latex allergy or anaphylaxis, is assessed. Interpreting the information provided by molecular allergy diagnosis needs a structured approach. It is necessary to evaluate single positivities and negativities, but also to appraise "the big picture" with perspective.

RESUMEN

BACKGROUND: An in vitro procedure based on a microarray containing many different allergen components has recently been introduced for use in allergy diagnosis. Recombinant and highly purified allergens belonging to different allergenic sources (inhalants, food, latex and hymenoptera) are present in the array. These components can either be genuine or cross-reactive, resistant or susceptible to heat and low pH, and innocuous or potentially dangerous. A large number of complex and heterogeneous relationships among these components has emerged, such that sometimes these interactions cannot be effectively managed by the allergist. In the 1960s, specialized languages and environments were developed to support the replacement of human experts with dedicated decision-making information systems. Currently, expert systems (ES) are advanced informatics tools that are widely used in medicine, engineering, finance and trading. METHODS: We developed an ES, named Allergenius ®, to support the interpretation of allergy tests based on microarray technology (ImmunoCAP ISAC ®). The ES was implemented using Flex, a LPA Win-Prolog shell. Rules representing the knowledge base (KB) were derived from the literature and specialized databases. The input data included the patient's ID and disease(s), the results of either a skin prick test or specific IgE assays and ISAC results. The output was a medical report. RESULTS: The ES was first validated using artificial and real life cases and passed all in silico validations. Then, the opinions of allergists with experience in molecular diagnostics were compared with the ES reports. The Allergenius reports included all of the allergists' opinions and considerations, as well as any additional information. CONCLUSIONS: Allergenius is a trustable ES dedicated to molecular tests for allergy. In the present version, it provides a powerful method to understand ISAC results and to obtain a comprehensive interpretation of the patient's IgE profiling.