Two hours later, IgG was detected using a rabbit anti-rat IgG- HRP conjugate (12500). fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope conversation. Conclusions/Significance The identification of glutamic acid residues with critical roles in IgE-binding to Ric SMO c Bisdemethoxycurcumin 3 and Ric c 1 support the potential use of free amino acids in allergy treatment. Introduction Castor bean (L.) contains approximately 50% oil, which has special characteristics such as a high viscosity, a high stability under heat and pressure, a low freezing point, and the ability to form waxy substances upon chemical treatment. As 0energy demands increase and Bisdemethoxycurcumin fossil fuels are limited, the development of alternative renewable fuels becomes imperative. Interest in biodiesel has been increasing owing to its environmental benefits and renewability [1]C[3]. As castor beans are a good biofuel source [3], castor bean cultivation is likely to increase, posing a risk of exposure to pollen allergens [4]C[6]. In previous studies, major castor bean allergens were identified [7]C[11]. We have recently reported the identification of IgE-binding epitopes of castor bean seed allergens, defining four continuous epitopes in Ric c 3 and two in Ric c 1 [12]. In the present study we identify critical amino acids for IgE binding and investigate cross-reactivity with allergens typically used for allergy diagnosis. Initially, we utilized the glutamic acid-specific Woodward’s Reagent K, WRK, (by dot blotting. After the first evaluation, patient serum with high intensity recognition of castor bean allergens was used in subsequent assays. For dot blot assays, 2S albumin or synthetic peptide (10 g in 10 L/dot) was spotted onto a nitrocellulose membrane and allowed to dry. The nitrocellulose membrane was incubated with total serum (150) or affinity supernatant or eluted fractions, FE and FG, from human or rat serum. Secondary anti-rat IgG or anti-human biotin IgE (0.5 mg/mL) (both diluted 12000) was then added to the membrane for 1 hour. Two hours later, IgG was detected using a rabbit anti-rat IgG- HRP conjugate (12500). For IgE detection, the membrane was subsequently incubated with streptavidin-biotinylated HRP complex for 1 h. The colour of all probes was developed with a substrate mixture: 5 mg of DAB in 4.9 mL of water, 300 L of 0.1 M imidazole, 100 L of Tris-HCl 2 M buffer Bisdemethoxycurcumin (pH 7.5) and 5 L of 30% H2O2. Rat peritoneal mast cells Wistar rats were obtained from the animal facility of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). All experimental procedures were approved by the animal research ethics board of the UENF (Proc. CEUA-UENF/112). Rats (weighing 250 g) were euthanized with CO2 and a peritoneal wash was performed by injection of 20 mL of DMEM (Dulbecco’s Modified Eagle Medium) made up of 12 U/mL of heparin. The abdomen was gently massaged for approximately 90 s. The peritoneal cavity was carefully opened and the fluid made up of peritoneal cells was aspirated with a Pasteur pipette. Thereafter, the Bisdemethoxycurcumin cells were transferred to Petri plates and incubated for 30 min at 37C. Two-thirds of the supernatant was aspirated and discarded. The mast cell-rich supernatant (1.8105 mast cells/mL) was separated into 100 L aliquots and kept at room temperature. Mast cell degranulation assays and cross-reactivity Rat peritoneal mast cells (100 L) were incubated with pre-immune serum (control) and activated for 60 min at 37C using 2S albumin polyclonal anti-rat IgE (2S alb AR IgE). After sensitization with 2S alb AR IgE, cells were washed twice with DMEM. Each experiment was carried out in the presence or absence of the synthetic peptides and a 2S albumin pool (100 ng). After incubation with antibodies and potential allergens (synthetic peptides, 2S albumin), histamine contents were determined (see below) and the cells (in 10 L) were stained for 15 min with 10 L of a solution made up of 0.1% toluidine blue, 10% formaldehyde and 1% acetic acid, pH 2.8, allowing the visualization of degranulated mast cells. Granulated.