A Novel Method for Total IgE Purification from Human Serum.

For Ab purification, high-affinity chromatography is commonly used. This technique results in high-purity Abs, but it requires highly specific knowledge and equipment. Commercial kits for purification of IgE are not available. Therefore, we established a (to our knowledge) novel method for the purification of total IgE from human serum. Sera from 19 allergic and nonallergic patients were included. After depletion of polyclonal IgG, total serum IgE was captured using anti-human IgE Abs coupled to beads, eluted from the beads, and incubated with protein G-coupled beads to increase the final purity. Purity analysis and Ab detection were performed by Western blot. Total serum IgE and purified IgE concentrations were analyzed using ELISA. To determine their functionality, primary human mast cells were sensitized with purified IgE and activated with anti-IgE or a relevant allergen. CD63+ expression and histamine release were used as readout parameters. Concentrations of purified total IgE corresponded with the levels of total serum IgE. Minor fractions of IgE remained attached to the beads, confirming an effective elution of IgE Abs. Only minimal amounts of IgG were found in the purified IgE fractions, confirming a high purity of IgE. Mast cells sensitized with purified IgE and subsequent activation with anti-IgE Ab or a relevant allergen showed increased expression of CD63+ and increased histamine release. This (to our knowledge) novel method represents a highly effective and widely accessible approach for purification of human serum IgE, which can improve the use of IgE-based in vivo and in vitro models and contribute to allergy research.

Autoreactive T cells and their role in atopic dermatitis.

Atopic dermatitis (AD) is an itchy, non-contagious relapsing and chronic inflammatory skin disease that usually develops in early childhood. This pathology is associated with food allergy, allergic asthma, allergic rhinitis and anaphylaxis which may persist in adulthood. The underlying mechanisms of AD (endotypes) are just beginning to be discovered and show a complex interaction of various pathways including skin barrier function and immune deviation. Immune reactions to self-proteins (autoantigens) of the skin have been identified in patients with inflammatory skin diseases, such as chronic spontaneous urticaria, connective tissue disease, pemphigus vulgaris and bullous pemphigoid. IgE antibodies and T cells directed against epitopes of the skin were observed in adult patients with severe and chronic AD as well. This was associated with disease severity and suggests a progression from allergic inflammation to severe autoimmune processes against the skin. IgE-mediated autoimmunity and self-reactive T cells might accelerate the ongoing skin inflammation or might contribute to the relapsing course of the disease. However, to date, the exact mechanisms of IgE-mediated autoimmunity and self-reactive T cells in the pathophysiology of AD are still unclear. The aim of this review is to evaluate the development of (autoreactive) T cells and their response to (auto)antigens, as well as the role of the peripheral tolerance in autoimmunity in the pathophysiology of AD, including the unmet needs and gaps.