RESUMEN
Ara h 2, a peanut 2S albumin, is associated with severe allergic reactions, but a homologous protein, soybean 2S albumin, is not recognized as an important allergen. Structural difference between these proteins might explain this clinical discrepancy. Therefore, we mapped sequential epitopes and compared the structure of Ara h 2, Soy Al 1, and Soy Al 3 (Gly m 8) to confirm whether structural differences account for the discrepancy in clinical responses to these two proteins. Commercially synthesized peptides covering the full length of Ara h 2 and two soybean 2S albumins were analyzed by peptide microarray. Sera from 10 patients with peanut and soybean allergies and seven non-atopic controls were examined. The majority of epitopes in Ara h 2 identified by microarray are consistent with those identified previously. Several regions in the 2S albumins are weakly recognized by individual sera from different patients. A comparison of allergenic epitopes on peanut and soybean proteins suggests that loop-helix type secondary structures and some amino acids with a large side chain including lone electron pair, such as arginine, glutamine, and tyrosine, makes the peptides highly recognizable by the immune system. By utilizing the peptide microarray assay, we mapped IgE epitopes of Ara h 2 and two soybean 2S albumins. The use of peptide microarray mapping and analysis of the epitope characteristics may provide critical information to access the allergenicity of food proteins.
Asunto(s)
Albuminas 2S de Plantas/inmunología , Epítopos/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Alérgenos/inmunología , Mapeo Epitopo , Humanos , Proteínas de Soja/inmunología , Glycine max/química , Glycine max/inmunologíaRESUMEN
BACKGROUND: Oral immunotherapy (OIT) with cow's milk (CM) has been reported to induce a number of specific antibody responses, but these remain to be fully characterized. Our objective was to explore whether IgE and IgG4 epitope binding profiles could predict the risk of side effects during CM OIT. METHODS: The study population consisted of 32 children (6-17 yr of age) with CM allergy: 26 children who successfully completed OIT and six children who discontinued therapy due to adverse reactions. We investigated sera drawn before and after OIT. We analyzed specific IgE and IgG4 binding to CM protein-derived peptides with a microarray-based immunoassay. Antibody binding affinity was analyzed with a competition assay where CM proteins in solution competed with peptides printed on the microarray. RESULTS: IgE binding to CM peptides decreased and IgG4 binding increased following the OIT in children who attained desensitization. Compared with children who successfully completed OIT, those who discontinued OIT due to adverse reactions developed increased quantities and affinity of epitope-specific IgE antibodies and a broader diversity of IgE and IgG4 binding, but less overlap in IgE and IgG4 binding to CM peptides. CONCLUSIONS: Detailed analysis of IgE and IgG4 binding to CM peptides may help in predicting whether CM OIT will be tolerated successfully. It may thus improve the safety of the therapy.
Asunto(s)
Desensibilización Inmunológica , Epítopos/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad a la Leche/terapia , Adolescente , Niño , Humanos , Resultado del TratamientoRESUMEN
Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cow's milk allergen ß-casein (ß-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. ß-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: ß-CN (57-68) and ß-CN (82-93), which matched fragments released by ß-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.
Asunto(s)
Caseínas/metabolismo , Digestión , Hipersensibilidad a los Alimentos/metabolismo , Jugo Gástrico/metabolismo , Inmunoglobulina E/inmunología , Leche/metabolismo , Péptidos/inmunología , Saliva/metabolismo , Alérgenos/química , Alérgenos/inmunología , Alérgenos/metabolismo , Animales , Caseínas/química , Caseínas/inmunología , Bovinos , Hipersensibilidad a los Alimentos/inmunología , Humanos , Leche/química , Leche/inmunología , Modelos Biológicos , Péptidos/química , Péptidos/metabolismoRESUMEN
SCOPE: IgE-epitope mapping of allergens reveal important information about antigen components involved in allergic reactions. The peptide-based microarray immunoassay has been used to map epitopes of some food allergens. We developed a peptide microarray immunoassay to map allergenic epitopes in parvalbumin from Atlantic cod (Gad m 1), the most consumed cod species in Spain. METHODS AND RESULTS: Sera from 13 fish-allergic patients with specific IgE to cod parvalbumin were used. A library of overlapping peptides was synthesized, representing the primary sequence of Gad m 1. Peptides were used to analyze allergen-specific IgE antibodies in patient sera. 100% of the patients recognized one antigenic region of 15 amino acids in length in Gad m 1. This region only partially correlated with one of the three antigenic determinants of Gad c 1 (Allergen M), parvalbumin from Baltic cod (Gadus callarias). In the 3D model of the protein, this region was located on the surface of the protein. CONCLUSION: We have identified a relevant antigenic region in Gad m 1. This epitope could be considered as a severity marker and provides additional information to improve fish allergy diagnosis and the design of safe immunotherapeutic tools.
Asunto(s)
Alérgenos/química , Mapeo Epitopo/métodos , Epítopos/química , Gadus morhua/inmunología , Inmunoglobulina E/sangre , Adolescente , Adulto , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos/sangre , Antígenos/inmunología , Estudios de Casos y Controles , Niño , Epítopos/inmunología , Femenino , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoensayo , Masculino , Datos de Secuencia Molecular , Parvalbúminas/química , Parvalbúminas/inmunología , Conformación Proteica , España , Población Blanca , Adulto JovenRESUMEN
BACKGROUND: Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3. OBJECTIVE: We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. METHODS: Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described. RESULTS: At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 µg/mL (interquartile range [25% to 75%], 0.1-0.43 µg/mL) at baseline to 10.5 µg/mL (interquartile range [25% to 75%], 3.95-45.48 µg/mL; P < .0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P < .0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2. CONCLUSION: OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.
Asunto(s)
Alérgenos/inmunología , Arachis/inmunología , Desensibilización Inmunológica , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad al Cacahuete/inmunología , Hipersensibilidad al Cacahuete/terapia , Administración Oral , Adolescente , Alérgenos/administración & dosificación , Afinidad de Anticuerpos/inmunología , Niño , Preescolar , Epítopos/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , LactanteRESUMEN
BACKGROUND: Food Allergy Herbal Formula-2 (FAHF-2) prevents anaphylaxis in a murine model of peanut allergy. Multiple food allergies (MFA) are common and associated with a higher risk of anaphylaxis. No well-characterized murine model of sensitization to multiple food allergens exists, and no satisfactory therapy for MFA is currently available. OBJECTIVE: To determine the effect of FAHF-2 in a murine model of MFA. METHODS: C3H/HeJ mice were orally sensitized to peanut, codfish, and egg concurrently. Oral FAHF-2 treatment commenced 1 day after completing sensitization and continued daily for 7 weeks. Mice were subsequently orally challenged with each allergen. RESULTS: Antibodies in sera from mice simultaneously sensitized with peanut, codfish, and egg recognized major allergens of all 3 foods, demonstrating sensitization to multiple unrelated food allergens (MFA mice). Sham-treated MFA mice exhibited anaphylactic symptoms accompanied by elevation of plasma histamine and hypothermia. In contrast, FAHF-2-treated MFA mice showed no anaphylactic symptoms, normal body temperature, and histamine levels after challenge with each allergen. Protection was accompanied by reduction in allergen-specific immunoglobulin E levels. Allergen-stimulated Th2 cytokine interleukin-4 and interleukin-13 production levels decreased, whereas the Th1 cytokine interferon-γ levels were elevated in cultured splenocytes and mesenteric lymph node cells in FAHF-2-treated mice. CONCLUSION: We established the first murine model of MFA. FAHF-2 prevents peanut, egg, and fish-induced anaphylactic reactions in this model, suggesting that FAHF-2 may have potential for treating human MFA.
Asunto(s)
Anafilaxia/prevención & control , Hipersensibilidad al Huevo/prevención & control , Peces/inmunología , Hipersensibilidad a los Alimentos/prevención & control , Hipersensibilidad al Cacahuete/prevención & control , Extractos Vegetales/uso terapéutico , Alérgenos/administración & dosificación , Alérgenos/efectos adversos , Alérgenos/inmunología , Anafilaxia/etiología , Anafilaxia/inmunología , Animales , Arachis/inmunología , Modelos Animales de Enfermedad , Hipersensibilidad al Huevo/inmunología , Femenino , Hipersensibilidad a los Alimentos/etiología , Hipersensibilidad a los Alimentos/inmunología , Humanos , Ratones , Ratones Endogámicos C3H , Hipersensibilidad al Cacahuete/inmunología , Extractos Vegetales/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Peanut allergy is relatively common, typically permanent, and often severe. Double-blind, placebo-controlled food challenge is considered the gold standard for the diagnosis of food allergy-related disorders. However, the complexity and potential of double-blind, placebo-controlled food challenge to cause life-threatening allergic reactions affects its clinical application. A laboratory test that could accurately diagnose symptomatic peanut allergy would greatly facilitate clinical practice. OBJECTIVE: We sought to develop an allergy diagnostic method that could correctly predict symptomatic peanut allergy by using peptide microarray immunoassays and bioinformatic methods. METHODS: Microarray immunoassays were performed by using the sera from 62 patients (31 with symptomatic peanut allergy and 31 who had outgrown their peanut allergy or were sensitized but were clinically tolerant to peanut). Specific IgE and IgG(4) binding to 419 overlapping peptides (15 mers, 3 offset) covering the amino acid sequences of Ara h 1, Ara h 2, and Ara h 3 were measured by using a peptide microarray immunoassay. Bioinformatic methods were applied for data analysis. RESULTS: Individuals with peanut allergy showed significantly greater IgE binding and broader epitope diversity than did peanut-tolerant individuals. No significant difference in IgG(4) binding was found between groups. By using machine learning methods, 4 peptide biomarkers were identified and prediction models that can predict the outcome of double-blind, placebo-controlled food challenges with high accuracy were developed by using a combination of the biomarkers. CONCLUSIONS: In this study, we developed a novel diagnostic approach that can predict peanut allergy with high accuracy by combining the results of a peptide microarray immunoassay and bioinformatic methods. Further studies are needed to validate the efficacy of this assay in clinical practice.
Asunto(s)
Inmunización , Hipersensibilidad al Cacahuete/diagnóstico , Hipersensibilidad al Cacahuete/inmunología , Albuminas 2S de Plantas/inmunología , Albuminas 2S de Plantas/metabolismo , Adolescente , Antígenos de Plantas/inmunología , Antígenos de Plantas/metabolismo , Niño , Preescolar , Biología Computacional/métodos , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina E/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Proteínas de la Membrana , Modelos Biológicos , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Valor Predictivo de las Pruebas , Análisis por Matrices de ProteínasRESUMEN
BACKGROUND: IgE epitope mapping of allergens reveals important information about antigen elicitors involved in allergic reactions. The peptide-based microarray immunoassay offers an advantage of scale and parallel design over previous methods of epitope mapping. It has been used to map epitopes of some food allergens but has never been used with fish allergens. OBJECTIVE: We sought to develop a peptide microarray immunoassay to map allergenic fish epitopes of two isoforms of Atlantic salmon (Salmo salar) parvalbumin, Sal s 1 beta 1 and Sal s 1 beta 2. METHODS: Sera from 16 fish-allergic patients with specific IgE to salmon parvalbumin were used. Twelve healthy volunteers were used as negative controls. A library of overlapping peptides was synthesized commercially, representing the primary sequence of Sal s 1 beta 1 and Sal s 1 beta 2. Peptides were used to analyze allergen-specific IgE antibodies by immunolabeling with patient sera. RESULTS: Three antigenic regions, not previously described, were identified in Sal s 1 beta 1. Two of them correlated with those previously reported in Gad c 1, parvalbumin from Baltic cod (Gadus callarias). No allergenic regions were found in Sal s 1 beta 2. This could be explained by crucial amino acid substitutions between isoforms. CONCLUSIONS: We have identified three antigenic regions in Sal s 1 beta 1 using a peptide microarray immunoassay. These three sequential epitopes formed a unique antigenic determinant in the three-dimensional model of the protein. In addition, we proved that isoforms from the same protein might have a different allergenic behavior.
Asunto(s)
Alérgenos/inmunología , Mapeo Epitopo/métodos , Inmunoensayo , Análisis por Micromatrices , Péptidos/inmunología , Salmo salar/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Niño , Epítopos/química , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Adulto JovenRESUMEN
BACKGROUND: Peanut allergy is a frequent and potentially life-threatening food allergy. Despite the large taxonomic distance between the plants, peanut-allergic patients often react to tree nuts such as walnuts. While the allergens of peanut and walnut have a high degree of homology in their amino-acid sequences, it is unknown whether this similarity is responsible for the observed co-reactivity. Therefore, we analyzed the binding of specific IgE antibodies to sequential epitopes of peanut and walnut in peanut-allergic patients with and without walnut allergy. METHODS: The IgE binding to previously described sequential epitopes of peanut and the homologous regions of walnut was assessed in 32 peanut-allergic patients using a peptide microarray technology. Twelve patients had a clinically relevant walnut allergy and 20 were tolerant to walnut. Inhibition assays with peanut peptides and corresponding walnut sequences were performed to show specific binding to sequential epitopes. RESULTS: No differences in the recognition of sequential epitopes could be found between peanut-allergic patients with or without walnut allergy. Only a few patients showed IgE binding to walnut sequences that corresponded to sequential epitopes of peanut. In the inhibition assays, no relevant cross-reacting IgE antibodies could be detected for the peptides analyzed. CONCLUSION: Our results indicate that although they share a rather high degree of homology with the corresponding regions of walnut allergens, the sequence stretches previously identified as sequential IgE binding epitopes of Ara h 1, Ara h 2 and Ara h 3 have no IgE binding equivalents in walnut allergens.
Asunto(s)
Alérgenos/inmunología , Antígenos de Plantas/inmunología , Epítopos/inmunología , Inmunoglobulina E/inmunología , Juglans/inmunología , Hipersensibilidad a la Nuez/inmunología , Hipersensibilidad al Cacahuete/inmunología , Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/inmunología , Adolescente , Adulto , Alérgenos/química , Antígenos de Plantas/química , Niño , Preescolar , Estudios de Cohortes , Mapeo Epitopo , Epítopos/química , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Proteínas de la Membrana , Hipersensibilidad a la Nuez/complicaciones , Hipersensibilidad al Cacahuete/complicaciones , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Análisis por Matrices de Proteínas , Adulto JovenAsunto(s)
Hipersensibilidad al Huevo/inmunología , Vacunas contra la Influenza/química , Ovalbúmina/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Ovalbúmina/efectos adversos , Ovalbúmina/inmunologíaRESUMEN
BACKGROUND: Lentils are often responsible for allergic reactions to legumes in Mediterranean children. Although the primary sequence of the major allergen Len c 1 is known, the location of the IgE-binding epitopes remains undefined. OBJECTIVE: We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. METHODS: One hundred thirty-five peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 patients with lentil allergy and 15 nonatopic control subjects by means of microarray immunoassay. Lentil-specific IgE levels, skin prick test responses, and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above interslide and intraslide cutoffs and confirmed by using inhibition assays with a peptide from the respective region. Hierarchic clustering of microarray data was used to correlate binding patterns with clinical findings. RESULTS: The patients with lentil allergy specifically recognized IgE-binding epitopes located in the C-terminal region between peptides 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil-specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition, and respiratory symptoms. Modeling based on the 3-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. CONCLUSION: Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response.
Asunto(s)
Alérgenos/inmunología , Epítopos , Inmunoglobulina E/sangre , Lens (Planta)/inmunología , Proteínas de Almacenamiento de Semillas/inmunología , Adolescente , Alérgenos/genética , Niño , Preescolar , Epítopos/química , Femenino , Humanos , Hipersensibilidad Inmediata/genética , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Lens (Planta)/genética , Masculino , Análisis por Micromatrices , Modelos Moleculares , Proteínas de Almacenamiento de Semillas/genéticaRESUMEN
BACKGROUND: Results from large-scale epitope mapping with a peptide microarray have been shown to correlate with clinical features of milk allergy. OBJECTIVES: We sought to assess IgE and IgG4 epitope diversity and IgE affinity in different clinical phenotypes of milk allergy and identify informative epitopes that might be predictive of clinical outcomes of milk allergy. METHODS: Forty-one subjects were recruited from a larger study on the effects of ingesting heat-denatured milk proteins in subjects with milk allergy. Using food challenges, subjects were characterized as being clinically reactive to all forms of milk (n = 17), being tolerant to heated milk (HM) products (n = 16), or having outgrown their milk allergy (n = 8). Eleven healthy volunteers without milk allergy served as control subjects. A peptide microarray was performed by using the previously published protocol. RESULTS: Subjects with milk allergy had increased epitope diversity compared with those who outgrew their allergy. HM-tolerant subjects had IgE-binding patterns similar to those who had outgrown their allergy, but IgG4-binding patterns that were more similar to those of the allergic group. Binding to higher numbers of IgE peptides was associated with more severe allergic reactions during challenge. There was no association between IgG4 peptides and clinical features of milk allergy. Using a competitive peptide microarray assay, allergic patients demonstrated a combination of high- and low-affinity IgE binding, whereas HM-tolerant subjects and those who had outgrown their milk allergy had primarily low-affinity binding. CONCLUSIONS: Greater IgE epitope diversity and higher affinity, as determined by using the peptide microarray, were associated with clinical phenotypes and severity of milk allergy.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Epítopos/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad a la Leche/inmunología , Adolescente , Alérgenos/inmunología , Secuencia de Aminoácidos , Niño , Preescolar , Mapeo Epitopo , Femenino , Calor , Humanos , Masculino , Proteínas de la Leche/inmunología , Datos de Secuencia Molecular , Fenotipo , Análisis por Matrices de ProteínasRESUMEN
BACKGROUND: The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample. OBJECTIVE: We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens. METHODS: A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of alpha(s1)-casein, alpha(s2)-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay. RESULTS: Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (r > or = 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array. CONCLUSION: A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.
Asunto(s)
Alérgenos/inmunología , Mapeo Epitopo/métodos , Epítopos/inmunología , Hipersensibilidad a la Leche/inmunología , Proteínas de la Leche/inmunología , Análisis por Matrices de Proteínas , Secuencia de Aminoácidos , Animales , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Leche/inmunología , Hipersensibilidad a la Leche/diagnóstico , Datos de Secuencia Molecular , Péptidos/inmunología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
With the evolution of peptide synthesis techniques and microarray technology, it is now possible to map and characterize allergenic epitopes on a microarray platform. The peptide microarray-based immunoassay allows simultaneous analysis of thousands of target peptides using small volumes of diluted serum samples, and has a promising future to become a superior testing tool for aspects of food allergy diagnosis and prognosis, as well as for designing recombinant allergens for safe immunotherapy. Characterization of allergenic epitopes provides fundamental knowledge for understanding mechanisms of food allergy. This chapter describes in detail the development of a sensitive and reliable peptide microarray-based immunoassay. The information includes a comparison of different slide substrates, effect of buffers on spot morphology, performance of printing pins, immunolabeling detection systems with different levels of sensitivity, and suggested approaches to data analysis.
Asunto(s)
Alérgenos/inmunología , Mapeo Epitopo/métodos , Hipersensibilidad/diagnóstico , Análisis por Micromatrices/métodos , Péptidos/inmunología , Humanos , Hipersensibilidad/inmunología , Inmunoensayo/métodos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Peptide microarray analysis is a novel method that can provide useful information on the nature of specific allergies. OBJECTIVE: We sought to determine the specificity and diversity of IgE and IgG4 antibodies binding to sequential epitopes of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin by using a peptide microarray-based immunoassay. METHODS: A microarray immunoassay was performed with sera from 31 children with IgE-mediated milk allergy (16 with positive oral milk challenge results [ie, the reactive group] and 15 with negative oral milk challenge results [ie, the tolerant group]). A library of peptides, consisting of 20 amino acids (AAs) overlapping by 17 (3-offset), corresponding to the primary sequences of alpha(s1)-, alpha(s2)-, beta-, and kappa-caseins and beta-lactoglobulin was printed on epoxy-coated slides. A region was defined as an epitope if it was statistically associated with reactive groups and recognized by at least 75% of reactive patients. RESULTS: By using this method, a total of 10 epitopes were identified: alpha(s1), AAs 28 to 50, 75% reactive and 26.7% tolerant; alpha(s2), AAs 1 to 20, 75% reactive and 13.3% tolerant; AAs 13 to 32, 75% reactive and 26.7% tolerant; AAs 67 to 86, 75% reactive and 33.3% tolerant; and AAs 181 to 207, 75% reactive and 20% tolerant; beta-casein, AAs 25 to 50, 75% reactive and 33.3% tolerant, AAs 52 to 74, 81.3% reactive and 26.7% tolerant; and AAs 154 to 173, 75% reactive and 33.3% tolerant; beta-lactoglobulin, AAs 58 to 77, 81.3% reactive and 40% tolerant; and kappa-casein, AAs 34 to 53, 87.5% reactive and 40% tolerant. CONCLUSION: Several regions have been defined as epitopes, which showed differential recognition patterns between reactive and tolerant patients. Further studies are needed to validate the utility of this assay in clinical practice.
Asunto(s)
Caseínas/inmunología , Mapeo Epitopo , Epítopos/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Lactoglobulinas/inmunología , Hipersensibilidad a la Leche/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Preescolar , Femenino , Humanos , Inmunoensayo , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Masculino , Datos de Secuencia Molecular , Análisis por Matrices de ProteínasRESUMEN
BACKGROUND: Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. Few options are available for treatment, and avoidance is the only therapy recommended. OBJECTIVE: We sought to identify relevant crustacean allergens for use as diagnostic and safe immunotherapeutic agents for subjects with shellfish allergy. METHODS: Thirty-eight patients were selected with immediate allergic reactions to shrimp and increased shrimp-specific serum IgE levels. One-dimensional and 2-dimensional electrophoresis of shrimp extracts were followed by IgE immunoblotting. Protein identification was done with matrix-assisted laser desorption/ionization-mass spectrometry and Edman sequencing. A cDNA library was generated from white pacific shrimp (Litopenaeus vannamei) and screened with primers designed on the basis of internal sequences obtained from 2-dimensional tryptic digests. Full-length cDNA clones were isolated from the library and sequenced. Recombinant protein was expressed and tested with sera from patients with shrimp allergy. RESULTS: Immunoblotting demonstrated IgE binding to a 20-kDa shrimp protein by 21 (55%) of 38 sera. Tryptic digestion of the protein followed by matrix-assisted laser desorption/ionization-mass spectrometric analysis and Edman sequencing identified it as a myosin light chain (MLC). Screening of the shrimp cDNA library resulted in isolation of a novel protein cDNA. Open reading frame translation provided the amino acid sequence of a new allergenic shrimp protein with high similarity to Bla g 8 (cockroach MLC). Recombinant protein was recognized by 17 patients, confirming the allergenicity of shrimp MLC. CONCLUSIONS: We have identified and cloned a new major shrimp allergen, Lit v 3.0101, an MLC protein.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad a los Alimentos/inmunología , Inmunoglobulina E/inmunología , Cadenas Ligeras de Miosina/inmunología , Penaeidae/inmunología , Mariscos/efectos adversos , Adolescente , Adulto , Alérgenos/genética , Animales , Niño , Femenino , Hipersensibilidad a los Alimentos/sangre , Biblioteca de Genes , Humanos , Inmunoglobulina E/sangre , Masculino , Cadenas Ligeras de Miosina/genética , Penaeidae/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
BACKGROUND: Better understanding of the relationship between antibody response to peanut and clinical sensitivity might lead to more accurate prognostication. OBJECTIVE: We sought to investigate peanut-specific IgE and IgG4 epitope diversity in relation to challenge-defined clinical sensitivity to peanut in a group of peanut-sensitized children. METHODS: Clinical sensitivity was determined by means of double-blind, placebo-controlled peanut challenges in 24 sensitized children. Six atopic control subjects were included. Specific IgE and IgG4 binding to 419 overlapping 15-amino-acid peptides representing the sequence of recombinant Ara h 1, Ara h 2, and Ara h3 was analyzed by means of microarray immunoassay. RESULTS: Peanut-sensitized patient sera bound significantly more IgE and IgG4 epitopes than control sera. This patient group reacted to the same Ara h 1, Ara h 2, and Ara h 3 epitopes as reported previously. There was a positive correlation between IgE epitope diversity (ie, number of epitopes recognized) and clinical sensitivity (r = 0.6), such that patients with the greatest epitope diversity were significantly more sensitive than those with the lowest diversity (P = .021). No specific epitopes were associated with severe reactions to peanut. IgG4 binding was observed to largely similar epitopes but was less pronounced than IgE binding and did not relate to the clinical sensitivity to peanut. IgE and IgG4 epitope-recognition patterns were largely stable over a 20-month period. CONCLUSION: Clinical sensitivity, as determined by means of double-blind, placebo-controlled peanut challenge, is positively related to a more polyclonal IgE response, which remains stable over time.
Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito B/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad al Cacahuete/inmunología , Adolescente , Especificidad de Anticuerpos , Arachis/química , Arachis/inmunología , Niño , Preescolar , Epítopos de Linfocito B/química , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/química , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Lactante , Masculino , Hipersensibilidad al Cacahuete/sangreAsunto(s)
Alérgenos/inmunología , Antígenos de Plantas/inmunología , Sesamum/inmunología , Alérgenos/química , Alérgenos/aislamiento & purificación , Secuencia de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/aislamiento & purificación , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/inmunología , Inmunoglobulina E/metabolismo , Datos de Secuencia Molecular , Semillas/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sesamum/químicaRESUMEN
BACKGROUND: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. OBJECTIVE: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. METHODS: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. CONCLUSION: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.