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3). Open in a separate window Figure 3 Proliferative responses of T cells from Cry j 2\immunized BALB/c, C57BL/6 and B10.S mice to Cry j 2, Cry j 1 and Cha o 1. and Cha o 1 share their B\cell epitope but not their T\cell epitope. This obtaining may provide a clue for the clarification of the T\cell and B\cell epitopes of Cry j 1 and Cha o 1, even though the data are influenced by H\2 complex restriction in mice. Considering that H\2 complex restriction affects cross responsiveness to Cry j 1 and Cha o 1 at the T\ and B\cell level in mice, we assessed the possible situation in humans uncovered sequentially to Japanese cedar pollen and Japanese cypress pollen. Introduction Pollinosis caused by Japanese cedar pollen (sugi in Japanese) is one of the commonest allergic respiratory diseases in Japan.1C3 In most patients with Japanese cedar pollinosis, it has been noted that symptoms persist long after the end of the pollen season for Japanese cedars (February to March).4,5 The Japanese cypress (hinoki in Japanese) pollen season runs from March to April and this pollen also causes pollinosis in Japan.4,6,7 Therefore, antigenicity or common antigenicity between Japanese cedar pollen and cypress pollen has been studied to seek the cause of the prolonged symptoms in cedar pollinosis patients. These studies indicated cross allergenicity between the two types of pollen based on serum levels of antibodies to the pollen or clinical scores.4,6C11 However, it is difficult to evaluate accurately the existence of common antigenicity as the cause of prolonged symptoms of Japanese cedar pollinosis by such studies, because patients could be sensitized to the two pollens independently. Two allergenic molecules of have been purified; Sugi basic protein (SBP; Cry j 1)12 and Cry j 2.13 Recently, the complete amino acid sequences of Cry j 1 and Cry j 2 were reported, with no homology being detected between the two antigens.14,15 In addition, purification and molecular cloning of a major allergenic molecule of [3H]thymidine incorporation assay. RPMI\1640 medium (Immuno\Biology Laboratories, Tokyo, Japan) supplemented with 1% normal mouse serum was used to suspend LNC and 8 105 cells were seeded into each well of 96\well plates (Nunc Maxisorp, Kamnstrup, Denmark) and cultured with 5 g of each antigen for 72 hr. The cultures were pulsed with 1 Ci of [3H]thymidine (ICN, Costa Mesa, CA) for the final 16 hr. Cells were harvested with a Labo Mash cell harvester (Laboratory Science Co., Ltd, Tokyo, Japan) and [3H]thymidine incorporated by the cells was determined by measurement of the radioactivity with a Beta\plate reader (Beckman Devices Inc., Fullerton, CA). Determination of antigen\binding specificity of serum antibody by Western blottingSerum collected from mice 10 days after immunization was used. The crude extract antigens (CEs) of Japanese cedar pollen and Japanese cypress pollen were prepared Triisopropylsilane as explained previously.12 Briefly, 10 l of 10 g/ml of Cry j 1, Cry j 2, Cha o 1, or Triisopropylsilane CE of Japanese cedar pollen and CE of Japanese cypress pollen were electrophoresed in 10% polyacrylamide gel in the presence of sodium dodecyl sulphate (SDSCPAGE). The proteins separated by SDSCPAGE were transferred to a polyvinylidenedifluoride membrane (Bio\Rad Laboratories, Hercules, CA). The membrane was blocked with 1% milk (Block Ace?, Dainippon Pharmaceutical Co., Osaka, Japan) for 1 hr at room temperature and then incubated with a 1 : 200 dilution of test mouse serum overnight at 4. After washing, to detect immunoglobulin G (IgG) antibody bound to the antigenic protein, the membrane Rabbit Polyclonal to AML1 was treated with a Vectastain Elite ABC Kit? (Vector Laboratories Inc., Burlingame, CA) made up of biotin\labeled anti\mouse IgG antibodies and streptavidinCperoxidase conjugate according to the manufacturers protocol. Finally, staining with 3\diaminobenzidine tetrahydrochloride (Nakarai Chemicals) was Triisopropylsilane performed for 1 min at room heat. Kaleidoscope Prestained Standard (Bio\Rad Laboratories) was.