Su S N, Shu P, Lau G X, Yang S Y, Huang S W, Lee Y C
Department of Medical Research, Veterans General Hospital Taipei, Taiwan, Republic of China.
J Allergy Clin Immunol. 1996 Sep;98(3):486-94. doi: 10.1016/s0091-6749(96)70080-5.
In a previous study we showed that antigen BG60 of Bermuda grass pollen contains isoallergens. Because the yield of purified isoallergens was low when a chromatofocusing technique was used, it was difficult to carry out further studies, such as determination of carbohydrate composition and structure.
The aim of this study was to establish a procedure to purify antigen BG60 proteins as a group and to characterize this group's physicochemical and immunologic properties.
A combination of chromatographic techniques (ion-exchange, gel filtration, blue gel affinity, and reverse-phase high-performance liquid chromatography) was used for the purification of BG60. Immunoblot and ELISA techniques were used to study BG60-specific IgE and IgG antibodies in patients' sera. The role of the carbohydrate moiety in antigenicity and allergenicity was examined with monoclonal antibodies and allergic sera by using periodate-treated BG60. Its carbohydrate composition was analyzed by high-performance anion-exchange chromatography with a pulsed amperometric detector.
Homogeneity of BG60 was demonstrated by a single sharp peak in reverse-phase high-performance liquid chromatography, a single band in sodium dodecylsulfate-polyacrylamide gel electrophoresis, and only one band stained by anti-BG60 monoclonal antibody. BG60-specific IgE and IgG antibodies were shown to be present in allergic sera. Six plant lectins were found to react with BG60. On periodate treatment, BG60 reduced binding toward its monoclonal antibody and human IgE and IgG. Carbohydrate composition analysis showed that BG60 contains three kinds of sugars: mannose, N-acetylglucosamine, and fucose (in a ratio of approximately 3:2:1) and a minute amount of xylose. The carbohydrate content is approximately 7.5%, and peptide content is about 92.5%.
A procedure was established for the purification of a large quantity of the BG60 antigen. The results suggest that the carbohydrate moiety of antigen BG60 may play an important role in the immune response.
在之前的一项研究中，我们发现百慕大草花粉的抗原BG60含有同种变应原。由于使用色谱聚焦技术时纯化同种变应原的产量较低，因此难以开展进一步研究，如确定碳水化合物组成和结构。
本研究旨在建立一种纯化抗原BG60蛋白组的方法，并对该蛋白组的理化和免疫特性进行表征。
采用多种色谱技术（离子交换、凝胶过滤、蓝色凝胶亲和和反相高效液相色谱）相结合的方法纯化BG60。采用免疫印迹和ELISA技术研究患者血清中BG60特异性IgE和IgG抗体。通过使用经高碘酸盐处理的BG60，利用单克隆抗体和过敏血清检测碳水化合物部分在抗原性和致敏性中的作用。采用带脉冲安培检测器的高效阴离子交换色谱法分析其碳水化合物组成。
反相高效液相色谱中的单一尖锐峰、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中的单一条带以及抗BG60单克隆抗体染色的仅一条带均证明了BG60的均一性。结果显示过敏血清中存在BG60特异性IgE和IgG抗体。发现六种植物凝集素可与BG60发生反应。经高碘酸盐处理后，BG60对其单克隆抗体以及人IgE和IgG的结合能力降低。碳水化合物组成分析表明，BG60含有三种糖：甘露糖、N-乙酰葡糖胺和岩藻糖（比例约为3:2:1）以及微量木糖。碳水化合物含量约为7.5%，肽含量约为92.5%。
建立了一种纯化大量BG60抗原的方法。结果表明，抗原BG60的碳水化合物部分可能在免疫反应中起重要作用。
