Performance of a component‐based allergen‐microarray in the diagnosis of cow's milk and hen's egg allergy

Background The food challenge test (FCT) is the gold standard for the diagnosis of food allergy. This procedure is time consuming, costly and can induce potentially severe symptoms. An ideal in vitro test should allow to avoid the FCT. Objective To assess the clinical performance of microarray for specific IgE (sIgE) detection in children with challenge‐proven/excluded cow's milk (CM) or hen's egg (HE) allergy. Methods One‐hundred and four children with suspected IgE‐mediated hypersensitivity to CM or HE were studied. In all patients, skin prick test, ImmunoCAP, microarray and FCT were performed. Results The microarray components Bos d 8 for CM (27/58 patients) and Gal d 1 (20/46 patients) and Gal d 2 (24/46) for HE were the most frequently recognized allergens. Using the FCT results as the reference parameter, sIgE to Bos d 8 and Gal d 1 had the highest area under the curves. These were not significantly different from those obtained using the ImmunoCAP. Use of 95% clinical decision points (CDP) for sIgE to Bos d 8 and Gal d 1 resulted in higher negative predictive values (78% and 79%, respectively) than those obtained with the ImmunoCAP (57% and 59%). Conclusions Our results show that in children with suspected CM or HE allergy, the microarray has a good ability to predict the FCT results. In a clinical application perspective, the microarray could be used as a second‐level assay, if the ImmunoCAP sIgE is <95% CDP. This approach would lead to a decrease in the number of the FCT to be performed, as well as of positive FCTs with a subsequent decrease in severe reaction risk. Cite this as: L. E. D'Urbano, K. Pellegrino, M. C. Artesani, S. Donnanno, R. Luciano, C. Riccardi, A. E. Tozzi, L. Ravà, F. De Benedetti and G. Cavagni, Clinical & Experimental Allergy, 2010 (40) 1561–1570.     

Minimal impact of extensive heating of hen's egg and cow's milk in a food matrix on threshold dose‐distribution curves

We analyzed reaction threshold data from 352 children undergoing open food challenges to hen's egg or cow's milk, either fresh or extensively heated into a muffin. There was no significant shift in dose‐distribution curves due to the baking process, implying that existing threshold data for these allergens can be applied to allergen risk management, even when these allergens are heat‐processed into baked foods.     

Hen's egg allergen in house and bed dust is significantly increased after hen's egg consumption—A pilot study

Environmental exposure to food allergens may be a risk factor for cutaneous sensitization. Previous studies could detect peanut allergen in house dust. In this pilot study, we wanted to investigate whether hen's egg allergen is detectable in house dust collected from different household areas and whether levels are increased after intentional hen's egg consumption. Hen's egg protein levels of dust samples were measured using ELISA. In 8 of 8 households, hen's egg was detectable in dust samples of eating area and bed. Forty‐eight hours after intentional hen's egg consumption, hen's egg protein levels were significantly increased in both. Still, further research is necessary to investigate whether hen's egg allergen in house and bed dust plays a role in sensitization via skin.     

Atopy patch tests are useful to predict oral tolerance in children with gastrointestinal symptoms related to non‐IgE‐mediated cow's milk allergy

Atopy patch tests (APTs) have been proposed for the diagnostic approach in children with non‐IgE‐mediated cow's milk allergy and gastrointestinal symptoms. We aimed to investigate the benefit of APTs in predicting oral tolerance in these patients. We prospectively evaluated 172 subjects with a sure diagnosis of non‐IgE‐mediated CMA and gastrointestinal symptoms (97 boys, 56.4%; age, 6.37 m; range, 2–12 m). At diagnosis, 113/172 (65.7%) children had positive APTs to cow's milk proteins (CMP). After 12 months of exclusion, diet APTs were repeated immediately before OFC. APTs significantly correlated (P < 0.001) with the OFC outcome (r 0.579). Diagnostic accuracy was sensitivity of 67.95%, specificity of 88.3%, PPV of 82.81%, NPV of 76.85%, and a +LR of 5.80. APTs are a valuable tool in the follow‐up of children with non‐IgE‐mediated CMA‐related gastrointestinal symptoms by contributing in determining whether an OFC can safely be undertaken.     

Screening for hen's egg and chicken meat specific IgE antibodies in Saudi patients with allergic disorders

Background

Allergy to hen''s egg and meat contributes significantly to the manifestations of food allergy all over the world.

Objectives

This study was performed to assess the presence of hen''s egg and meat specific IgE antibodies among patients investigated for various allergic disorders.

Methods

This is a retrospective study performed at King Khalid University Hosptial, Riyadh. Data from 421 patients with allergic disorders screened for food specific IgE antibodies between January 2009 and March 2011 were analyzed. Sixty (14.25%) patients including 42 males and 18 females with the mean age (sd) of 7.5 (7.4) years were found to have specific IgE antibodies against hen''s egg and chicken meat. There were 56 (93.3%) children and 4 (6.7%) adult patients. Specific IgE antibodies were measured by radioallergosorbent test (RAST) using Pharmacia ImmunoCAP 250 analyzer.

Results

Atopic dermatitis was the most common (55%) clinical condition. Out of the total 60 patients harboring hen''s egg and chicken meat specific IgE antibodies high levels of egg white, yolk and chicken meat specific IgEs were detected in 58 (96.6%), 37 (61.6%) and 6 (10%) patients respectively. Both the egg white and yolk antibodies coexisted in 35 (58.3%) patients.

Conclusion

Sensitization against hen''s egg was higher compared to the chicken meat. Egg white sensitization higher than the egg yolk particularly in Saudi children with food related allergic disorders.     

Intolerance to hydrolysed cow's milk proteins in infants: clinical characteristics and dietary treatment.

BACKGROUND: Multiple food intolerance in infants, including intolerance to extensively hydrolysed proteins (HP), is often difficult to treat. However, few data have been reported on clinical outcome and dietary treatment of these patients. AIMS: To evaluate the clinical characteristics of patients with HP-intolerance and the long-term outcome of treatment with ass' milk. PATIENTS AND METHODS: This study included 21 HP-intolerant infants (15 males, median age at diagnosis 2 months) treated with an ass' milk-based diet and 70 cow's milk (CM) intolerant infants (40 males, median age at diagnosis 3 months) treated with casein hydrolysate milk-based diet. All patients were followed-up for a median period of 4 years. Both HP-intolerance and intolerance to other foods were diagnosed according to the double-blind placebo-controlled procedure. Formal CM-challenges were conducted at yearly intervals until tolerance was demonstrated. At diagnosis and after one year of the respective diets, the following growth parameters were determined: relative weight for sex and age, relative weight for height and height z-score. RESULTS: During the study period, multiple food intolerance was documented in 21/21 HP-intolerant infants (ass' milk group) and in 20/70 infants with CM-intolerance but tolerating HP (casein hydrolysate group) (P < 0.0001). In the ass' milk group, the more frequent food intolerances were toward soya, oranges, tomatoes and fish; goat's milk intolerance was demonstrated in five out of six patients receiving this food, and sheep's milk derivatives intolerance in four out of seven; these patients tolerated ass' milk. During the study period 3/21 patients in the ass' milk group became ass' milk intolerant; they showed vomiting (one cases) or diarrhoea (two cases). A lower percentage (52%) of patients in the ass' milk group became CM-tolerant during the study period than in the casein hydrolysate group (78%) (P < 0.01) and the age of the children at CM-tolerance was higher in the ass' milk than in the casein hydrolysate-treated children (P < 0.05). At diagnosis, a higher frequency of cases with elevated serum total IgE and specific IgE to CM antigens (P < 0.01) was observed in the ass' milk group. No difference was recorded between the two treatment groups in any of the growth parameters considered either at diagnosis or during the follow-up. CONCLUSIONS: HP-intolerant patients showed a higher frequency of persistent food intolerance and of multiple food intolerance than patients tolerating casein hydrolysate. Ass' milk feeding was confirmed as a safe and valid treatment of the most complicated cases of multiple food intolerance.     

Biochemical markers of bone metabolism in children with cow's milk allergy

Introduction

Patients with cow''s milk allergy (CMA) and following a cow milk protein-free diet for a long time are potentially at risk of developing bone abnormalities. To assess the balance between bone formation and resorption processes, we determined serum concentrations of osteocalcin (OC), bone alkaline phosphatase (BALP), C-terminal telopeptide of type I collagen (CTX), fetuin-A, osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in children with CMA.

Material and methods

The study included 50 prepubertal children with diagnosed cow''s milk allergy, who were under systematic medical and nutritional care at the Institute of Mother and Child and 40 healthy counterparts as a control group. The concentrations of bone metabolism markers were determined by immunoenzymatic assays.

Results

The diets of all investigated children were correct in terms of phosphorus and magnesium contents but deficient in terms of calcium and vitamin D. Serum OC and CTX as well as fetuin-A concentrations were similar in both studied groups. The BALP activity was significantly (p < 0.05) higher in children with cow''s milk allergy than in the controls. Serum OPG concentration was comparable in both groups, but the RANKL level was higher (p < 0.05) in CMA children than in healthy ones. Hence, the ratio of OPG/RANKL was lower in children with CMA.

Conclusions

Our study demonstrates slight disturbances in the profile of bone metabolism markers in growing children with CMA. The increase in RANKL level and decrease in OPG/RANKL ratio may contribute to intensification of bone resorption in these patients.     

Removal of bovine serum albumin from cow's milk using chicken egg‐yolk antibodies immobilized on chitosan gel

Polyclonal chicken antibodies raised against bovine serum albumin (BSA) were immobilized on chitosan gel for the immunoaffinity isolation of BSA from cow's milk. Antibodies (IgY) against BSA were isolated from egg‐yolk, purified and antibody reactivity to antigen was measured. IgY developed against BSA was reduced by 2‐mercaptoethylamine. The reactivities of reduced and whole IgY against BSA were not significantly different. The reduced IgY was covalently linked to chitosan gel through stable covalent thioether linkages using sulfo‐succinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐l‐carboxylate (sulfo‐SMCC) as a cross‐linker. The density of antibody IgY immobilized on chitosan gel was approximately 3–5 mg per ml of chitosan gel. The ligand‐binding capacity of immobilized IgY towards BSA was 0.35–0.44 mg BSA per ml of chitosan gel. A single pass of skimmed milk through the column allowed the removal of BSA from the milk sample. The milk sample was analyzed, before and after immunoaffinity separation, by SDS‐PAGE. BSA was desorbed with 0.5 M‐glycine‐HCl buffer at pH 2.8 but the reusability of the column was limited to three cycles. Alternatively, BSA was desorbed with 0.5 M‐glycine‐HCl buffer containing 2 M‐NaCl at pH 4.6 after longer incubation times at a slower flow rate. The low ligand‐binding capacity was not an impedement to reuse of the column. The column was reused more than 20 times with minimal loss of binding capacity.     

Comparison of intestinal anaphylactic reactions in sensitized mice challenged with untreated bovine milk and homogenized bovine milk

Outbred NMRI mice were sensitized for high IgE production either by subcutaneous injections of low doses of untreated bovine milk or homogenized bovine milk in combination with intraperitoneal injections of Freund's Complete Adjuvant or by oral administration of untreated or homogenized bovine milk without adjuvant. When analysed in murine passive cutaneous anaphylaxis test both types of milk resulted in production of reaginic antibodies against bovine milk proteins when given subcutaneously. When given orally, homogenized milk resulted in reagin production in 10 out of 14 mice, whereas untreated milk resulted in reagin production in only one out of 12 mice. The sensitized mice, and control mice, were orally challenged with either untreated milk, homogenized milk or 0.9% NaCl. Examination of the intestines 40 min after oral administration revealed that homogenized milk, contrary to untreated milk or 0.9% NaCl, resulted in a large increase in the mass of the proximal gut segment of mice sensitized orally with homogenized milk compared with control mice orally challenged with saline (P less than 0.001), and only mice both sensitized and challenged orally with homogenized milk showed degranulation of mast cells in the intestinal wall. By contrast, subcutaneously sensitized mice or mice sensitized orally with untreated bovine milk showed no significant intestinal reaction upon oral challenge with either homogenized or untreated bovine milk. These observations may indicate that the route of sensitization is of great importance when intestinal reactions are to be studied, and that homogenization of bovine milk may render the milk more aggressive with respect to its ability to induce intestinal reactions. The study indicates that mice may be an attractive experimental animal model for mimicking the intestinal anaphylactic reactions of cow milk-allergic humans.     

Allergenicity of hen's egg‐white proteins: IgE binding of native and deglycosylated ovomucoid

Ovomucoid (OM) is a major allergen of hen's egg‐white. Carbohydrate moieties from the glycoprotein were removed by chemical deglycosylation. Deglycosylated ovomucoid (d‐OM) consists of five isoforms with molecular weights (MW) between 20.7 and 21.5 kDa whereas native OM has a MW of 28 kDa as determined by matrix‐assisted laser desorption/ionization time of flight mass spectrometry. The IgE‐binding properties of both proteins were investigated. All patients’ sera tested, showed strong IgE binding to both native OM and d‐OM in SDS‐PAGE/immunoblot. In enzyme‐allergosorbent test experiments, 50% inhibition of IgE binding was observed, at almost the same inhibitor concentrations of 67 and 57 ng ml^‐1 for native OM and d‐OM respectively. Results indicate that only epitopes on the protein backbone are responsible for IgE binding while carbohydrate residues do not participate in allergenic structures of OM.