Aplicación de la tecnología microarray al manejo diagnóstico de la alergia a proteínas de leche de vaca

Resumo

BACKGROUND; Cow’s milk allergy (CMA) is a common and often transitory disease affecting approximately 2.5% of children less than 2 years of age. The mainstay of treating milk allergy consists of prescribing a milk-free diet and having the patient return for regular follow-up visits to reevaluate the status of their allergy. The majority of children with IgE-mediated milk allergy become tolerant. At the outset, the difficulty lies in predicting which children may develop severe allergic reactions to milk and which will remain allergic for a lifetime. Different cutoff values for skin prik test (SPT) and specific IgE antibodies levels have been previously given to predict the result of the food challenge test, in cows’ milk allergic children. Peptide microarray analysis is one method that may provide useful information on the nature of specific allergies. OBJETIVE: The aims of this study is evaluate the accuracy of previously proposed cut-off values in our population of allergic patients and to determine the IgG4 and IgE specificity and diversity to sequential epitopes of as1-, as2-, b-, and a-caseins, and a-lactoglobulin using a peptide microarray-based immunoassay. METHODS: A transversal study which included children 0 to 14 years old refered to the Allergy Clinic of Ramón y Cajal Hospital and diagnosed cows’ milk allergy: history of allergic reaction after ingestion of milk and positive SPT and/or positive specific IgE antibodies determination. After six months of milk-free diet, a complete anamnesis, SPT with cow’s milk, alfalactoalbumin, betalactoglobuline and casein, specific IgE antibodies were determinated for the same allergens. Simpled-blind controlled food challenges (SBCFC) with previous authorization of the parents were performed in those children with specific IgE antibodies determination lower than 2.5 KU/l. In a posteror step, a microarray immunoassay was performed using sera from 31 IgE-mediated milk allergic children (16 positive to oral milk challenge, “reactive group”, and 15 negative, “tolerant group”). A library of peptides, consisting of 20 amino acids overlapping by 17 (3-offset), corresponding to the primary sequences of as1-, as2-, b-, and a-caseins, and a-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: 162 children were included in the study. Only 85 children had specific IgE lower than 2.5KU/l and an oral challenge (SBCFC) were performed. 54 tolerate (63.5%). Best cutoff points for SPT to milk and fractions were 3mm. Mapping milk allergens utilizing a peptide microarray assay, a total of 10 epitopes were identified: - Alpha s1: AA 28-50 reactive 75%, tolerant 26.7% (p=0.012). - Alpha s2: AA 1-20 reactive 75%, tolerant 13.3% (p=0.001); AA 13-32 reactive 75%, tolerant 26.7% (p=0.012), AA 67-86 reactive 75% tolerant 33.3% (p=0.032) and AA 181-207 reactive 75% tolerant 20% (p=0.004). - Beta-casein: AA 25-50 reactive 75% tolerant 33.3% (p=0.032), AA 52-74 reactive 81.3% tolerant 26.7% (p=0.004) and AA 154-173 reactive 75% tolerant 33.3% (p=0.032). - Beta-lactoglobulin: AA 58-77 reactive 81.3% tolerant 40% (p=0.029). - Kappa-casein: AA 34-53 reactive 87.5% tolerant 40% (p=0.009). CONCLUSION: Available diagnostic test in daily practice have limited utility in predicting the result of the oral challange. Several regions have been defined as epitopes, showing a differential recognition pattern between reactive and tolerant patients. Further studies are needed to validate the utility of this assay in clinical practice.