Comparable levels of pollen extracts were blotted onto nitrocellulose (28) and effective transfer was confirmed by Ponceau S staining from the membranes

Comparable levels of pollen extracts were blotted onto nitrocellulose (28) and effective transfer was confirmed by Ponceau S staining from the membranes. unfilled M13 phages (x-axis). Mean OD beliefs +/?SD (y-axis) match bound Ac-Lys-AMC anti-phage antibodies. NIHMS63816-product-01.pdf (1.1M) GUID:?B7D434A6-F3C0-4296-B0D7-E6A51F1FFD52 Abstract The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens having a molecular mass of ~25C30 kDa. Group 1 allergens are identified by 95% of grass pollen sensitive patients. We investigated the IgE acknowledgement of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollenCallergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1Cderived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed similar high-affinity binding to Phl p 1 like a total human being IgE-derived Ab realizing the allergens C terminus. In a set of surface plasmon resonance experiments simultaneous allergen acknowledgement of all three binders was shown. Actually in the presence of the three binders, sensitive individuals polyclonal IgE reacted with Phl p 1, indicating high-density IgE acknowledgement of the Phl p 1 allergen. Our results display that multiple IgE Abs can bind with high denseness to Phl p 1, which may clarify the high allergenic activity and sensitizing capacity of this allergen. The number of individuals suffering from allergic symptoms such as hay fever, asthma, allergic pores and skin diseases, food allergy, or severe anaphylactic reactions offers increased to nearly a third of Ac-Lys-AMC the population in industrialized countries (1). A major hallmark of allergic diseases is the formation of specific IgE Abdominal muscles against per se harmless environmental Ags called allergens. Owing to weighty and long term launch of pollen, grasses symbolize probably one of the most potent and frequent allergen sources worldwide (2, 3). Grass pollen Rabbit Polyclonal to Keratin 15 consists of several allergens with high allergenic activity and amazingly high stability to pH and heat variations. Among those, group 1 allergens are the most common and important (4, 5). In fact, one of the 1st pollen allergens ever purified was the group 1 allergen from rye grass (6). This allergen resource is also suspected to be responsible for the initial hay fever epidemic in northern Europe when grass pollen was identified as a cause of hay fever (7). Group 1 allergens represent glycoproteins having a molecular mass of ~30 kDa. They may be identified by 90% of grass pollenCallergic individuals and happen as highly cross-reactive allergens in pollen of most, if not all, grass species, including temperate and tropical grasses (8, 9). Phl p 1 from timothy grass pollen is one of the best characterized group 1 allergens (10). It has been demonstrated to contain the majority of group 1Cspecific IgE and T cell epitopes, and its three-dimensional structure has been solved by x-ray crystallography (11C14). Several clinical studies possess shown the high allergenic activity and medical relevance Ac-Lys-AMC of Phl p 1 (5, 15), and it can be used like a diagnostic marker for the recognition of grass pollenCallergic individuals (16) due to its strong cross-reactivity with group 1 allergens from related grass species as shown by Kahn and Marsh (17). A key role as a possible initiator of grass pollen allergy has recently been attributed to Phl p 1 based on the longitudinal analysis of IgE reactivity toward a panel of different grass pollen Ac-Lys-AMC allergens in Ac-Lys-AMC birth cohorts (18). Interestingly, Phl p 1 was the grass pollen allergen with the highest prevalence of IgE acknowledgement in early existence whereas sensitization to additional allergens occurred later on in existence (18). With this study we investigated whether the high allergenic activity of Phl p 1 can be attributed to its acknowledgement of IgE Abdominal muscles from sensitive individuals. IgE-derived single-chain variable fragments (ScFvs) specific for Phl p 1 were isolated by combinatorial cloning from a grass pollenCallergic patient. Binding sites for the IgE-derived ScFvs were mapped and visualized within the three-dimensional structure of the allergen. The simultaneous high-affinity binding of the IgE-derived ScFvs was demonstrated in surface plasmon resonance (SPR) experiments. Our study shows that Phl p 1 bears a high number of unique IgE epitopes providing a possible explanation for its high sensitizing potential and allergenic activity. Materials and Methods Allergens, Abs, sera from sensitive patients, and synthetic peptides Recombinant allergens were purchased from Biomay (Vienna, Austria). The Phl p 1Cspecific IgE Fab clone 25 was isolated from a combinatorial library in a earlier study (19) (Table I). The V regions of this clone were also grafted onto a complete human being monoclonal.