Development of hypoallergenic food product and in-HOUSE elisa to differentiate distinct severity of COW's milk allergy as biscotti, baked, fermented and pasteurized milk reactive and tolerant ones

Avoidance of culprit allergen is still the mainstay of treatment in food allergy. However, the difficulties such as adherence to elimination diet or accidental allergen exposure have increased the research in food allergy treatment in recent years. Oral immunotherapy (OIT) is a treatment model of food allergy, and it is defined as the administration of the allergen in slowly increasing doses at certain time intervals until the daily-consumed dose is reached. Although immunotherapy with baked milk products seems safe, anaphylaxis can occur in some patients. Therefore, developing a hypoallergenic product that has less allergenicity than baked milk would open a new horizon in the desensitization treatment of cow's milk allergy. Here, we mainly aimed to produce a more hypoallergenic product than conventional cake, develop indirect in-house ELISA to check the tolerance status with milk, cheese, and yogurt, to check IgE reactivity of patients' sera via western blotting, indirect in-house ELISA and basophil activation test (BAT). First, a hypoallergenic product candidate was developed by baking the cake twice, which was called "biscotti". The proteins were isolated from the new hypoallergenic product (biscotti), conventional baked milk (cake) and pasteurized milk and the total protein concentration was determined by Bradford assay. The protein content for each product was studied by SDS-PAGE and proteomics. Serum samples were collected from patients with milk allergy who applied to the Pediatric Allergy Division in Koc University School of Medicine. Milk specific IgE (sIgE) binding assays were performed by Western blotting, indirect in-house ELISA by using patients' sera and BAT by using patients' whole blood. In the band intensity analysis, the casein band intensity was observed lower in the hypoallergenic product candidate than in the conventional cake (p=0.014). Proteomics analysis showed that the intensities of each casein fraction and β-lactoglobulin were decreased as the cooking time increased in all milk products (biscotti 1h, biscotti 2h and biscotti 3h, conventional cake). The low binding capacity of milk sIgE to the newly developed hypoallergenic product compared to the conventional cake in the sera of children with milk allergy was shown first by western blotting (p=0.0012) and then by indirect in-house ELISA (p=0.0001). Biscotti supernatant was used as a stimulating allergen in BAT and the results indicated that it could be used for further experiment before oral food challenge test. In this study, the low allergenicity of the newly developed hypoallergenic product "biscotti" has been demonstrated by in vitro experiments. Our hypoallergenic product could be a safe and useful treatment option in patients with severe milk allergy who are reactive to baked milk. We aim to confirm the low allergenicity of this product by in vivo experiments (oral food challenge tests) and show the effects in tolerance induction in the further steps.