Ludmilla Bardina

Specificity of IgE antibodies to sequential epitopes of hen's egg ovomucoid as a marker for persistence of egg allergy

Background:  Approximately two‐thirds of egg‐allergic infants become tolerant within the first 5 years of life.

IgE and IgG Binding Epitopes on α-Lactalbumin and β-Lactoglobulin in Cow’s Milk Allergy

Background: Cow’s milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE and IgG binding epitopes for αs1-casein, a major cow’s milk allergen, and found an association between recognition of certain epitopes and clinical symptoms of cow’s milk allergy (CMA). Since α-lactalbumin (ALA) and β-lactoglobulin (BLG) are suspected to be significant allergens in cow’s milk, we sought to determine the structure of sequential epitopes recognized by IgE antibodies to these proteins. We further sought to assess the pattern of epitope recognition in association with the clinical outcome of CMA. Methods: According to the known amino acid sequence of ALA and BLG, 57 and 77 overlapping decapeptides (offset by two amino acids), respectively, were synthesized on a cellulose derivatized membrane. Sera from 11 patients 4–18 years of age with persistent CMA (IgE to cow’s milk >100 kUA/l) were used to identify IgE binding epitopes. In addition, 8 patients <3 years of age and likely to outgrow their milk allergy (IgE to cow’s milk <30 kUA/l) were used to investigate the differences in epitope recognition between patients with ‘persistent’ and those with ‘transient’ CMA. Seven patients 4–18 years of age were used for assessing the IgG binding regions. Results: In patients with persistent allergy, four IgE binding and three IgG binding regions were identified on ALA, and seven IgE and six IgG binding epitopes were detected on BLG. The younger patients that are likely to outgrow their allergy recognized only three of these IgE binding epitopes on BLG and none on ALA. Conclusions: The presence of IgE antibodies to multiple linear allergenic epitopes may be a marker of persistent CMA. The usefulness of IgE binding to distinct epitopes on whey proteins in defining the patients that would have a lifelong CMA needs to be investigated in further studies.

Humoral and cellular responses to cow milk proteins in patients with milk‐induced IgE‐mediated and non‐IgE‐mediated disorders

Background:  Cow milk allergy (CMA) is one of the most common food allergies in childhood. Patients with CMA present with a wide range of immunoglobulin (Ig)E‐ and non‐IgE‐mediated clinical syndromes. Limited information is known about the specific humoral and cellular responses to cow milk proteins in these various forms of CMA.

Peanut epitopes for IgE and IgG4 in peanut-sensitized children in relation to severity of peanut allergy

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.

Correlation of IgE/IgG4 milk epitopes and affinity of milk-specific IgE antibodies with different phenotypes of clinical milk allergy.

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.

Mapping of the IgE and IgG4 sequential epitopes of milk allergens with a peptide microarray–based immunoassay

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.

Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.

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.

Development of a novel peptide microarray for large-scale epitope mapping of food allergens

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.

A bioinformatics approach to identify patients with symptomatic peanut allergy using peptide microarray immunoassay.

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.

Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family.

Background IgE‐mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins.