Clinical manifestations and risk factors of anaphylaxis in pollen\food allergy syndrome. 1 (54.8% identity to Bet v 1), Que ac 2 (79.7% to Bet v 2), Que ac 3 (24.9% to Bet v 3), 6 (71.3% to Bet v 6), Que ac 7 (80.9% to Bet v 7), and Que ac 8 (78.9% to Bet v 8). Que ac 1 sIgE was the most frequently recognized (84.0%), followed by Que ac 2 (12.0%), Que ac 3 (6.0%), and three other allergens (2.0% each). Que ac 1 was a dominant allergen affecting 83.7% of patients suffering from allergic rhinoconjunctivitis, and 92.9% of pollen food allergy syndrome patients. Conclusion Five novel IgE reactive components of sawtooth oak were characterized using transcriptome analysis. Que ac 1 is the single most important component allergen of sawtooth oak pollen. Rosetta\2 (DE3) (Novagen). The expression was induced by adding 1?mM isopropyl\1\thio\\galactopyranoside. Recombinant Que ac 2 and 3 were purified from the Polaprezinc soluble fractions. Que ac 6, 7, and 8 were purified from the inclusion bodies using Ni\NTA resin (Qiagen), dialyzed against phosphate\buffered saline (PBS), pH 7.4, and stored at ?20C. Recombinant Que ac 1 was also produced by the same way as described previously. 6 In order to identify the recombinant proteins, they were confirmed by liquid chromatography\coupled electrospray ionization\tandem mass spectrometry (LC ESI MS/MS) after gel excision. In\gel tryptic digestion was performed for tandem mass spectrometry at ProteomeTech. 3.3. IgE reactivity of recombinant allergens Specific IgEs to the recombinant allergens were measured using the ImmunoCAP platform. For ImmunoCAP analysis, biotinylation was performed using EZ\link? Sulfo\NHS\LC\Biotin (ThermoFisher Scientific). Briefly, recombinant Que ac 2, 3, 7, and 8 (2?mg) were incubated with NHS\LC\biotin on ice for 4?h and then dialyzed extensively against PBS to remove unreacted NHS\LC\biotin. Biotinylated proteins were loaded onto streptavidin ImmunoCAPs. IgE antibody binding to the proteins was measured using the Phadia UniCAP 100 Rabbit polyclonal to ABCB5 system according to the manufacturer’s instructions. An IgE value 0.35?kU/L was considered positive. IgE reactivity to recombinant Que ac 6 was examined by ELISA, because it was not readily biotinylated due to aggregation after dialysis against PBS. The microtiter plates (Corning Inc.) were coated with recombinant Que ac 6 (2?g/ml in 50?mM sodium carbonate, pH 9.6) and incubated overnight at 4?C. After blocking with 3% skim milk in PBS containing 0.05% Tween 20 (PBST), serum samples (1:4 dilution in PBS) containing 1% bovine serum albumin were applied and incubated for one hour at room temperature. The IgE antibodies were detected by incubating biotinylated goat anti\human IgE (1:1000) (Vector) for one hour, followed by incubation with streptavidin peroxidase (1:1,000) (Sigma\Aldrich) for 30?min. Color was developed using Polaprezinc TMB Microwell Peroxidase Substrate (SeraCare Life Science, Inc.). The Polaprezinc absorbance at 450?nm was measured after the addition of 0.5?M H2SO4. All of the samples were duplicated. The mean absorbance plus double standard deviation of the negative controls was determined as a cutoff value. 4.?RESULTS 4.1. Identification of allergen homologues by transcriptome analysis We were able to identify 6 birch allergen homologous molecules (Table?2). Que ac 1 (GenBank accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”MN201198″,”term_id”:”1917169568″,”term_text”:”MN201198″MN201198), 2 (“type”:”entrez-nucleotide”,”attrs”:”text”:”MW506866″,”term_id”:”2044045898″,”term_text”:”MW506866″MW506866), 3 (MW50686), 6 (“type”:”entrez-nucleotide”,”attrs”:”text”:”MW506868″,”term_id”:”2044045903″,”term_text”:”MW506868″MW506868), 7 (“type”:”entrez-nucleotide”,”attrs”:”text”:”MW506869″,”term_id”:”2044045905″,”term_text”:”MW506869″MW506869), and 8 (“type”:”entrez-nucleotide”,”attrs”:”text”:”MW506870″,”term_id”:”2044045907″,”term_text”:”MW506870″MW506870) share 54.8% identity to Bet v 1, 79.7% to Bet v 2, 24.9% to Bet v 3, 71.3% to Bet v 6, 80.9% to Bet v 7, and 78.9% to Bet v 8, respectively. However, a Bet v 4 homologous molecule was not found. TABLE 2 Allergen homologues obtained from sawtooth oak pollen transcriptomes Rosetta\2 (DE3) (Figure?1). The molecular identity of the recombinant proteins was confirmed again using LC ESI MS/MS (Table?3). Que ac 1 was the most frequently recognized by serum IgE (84.0%), followed by Que ac 2 (12.0%), Que Polaprezinc ac 3 (6.0%), and the other three allergens (2.0% each). Only the Que ac Polaprezinc 2 allergen was recognized by serum IgE from two patients who were not sensitive to Que ac 1 and increased the diagnostic sensitivity by 4.0% (Table?4). None of the other allergens added diagnostic value to Que ac 1..