IMMUNOLOGICAL AND PHYSICOCHEMICAL STUDIES OF BERMUDA GRASS-POLLEN ANTIGEN BG60

Background: In a previous study we showed that antigen BG60 of Bermuda grass pollen contains isoallergens. Because the yield of purified iso allergens was low when a chromatofocusing technique was used, it was d ifficult to carry out further studies, such as determination of carboh ydrate composition and structure. Objective: The aim of this study was to establish a procedure to purify antigen BG60 proteins as a group a nd to characterize this group's physicochemical and immunologic proper ties. Methods: A combination of chromatographic techniques (ion-exchan ge, gel filtration, blue gel affinity, and reverse-phase high-performa nce liquid chromatography) was used for the purification of BG60. Immu noblot and ELISA techniques were used to study BG60-specific IgE and I gG antibodies in patients' sera. The role of the carbohydrate moiety i n antigenicity and allergenicity was examined with monoclonal antibodi es and allergic sera by using periodate-treated BG60. Its carbohydrate composition was analyzed by high-performance anion-exchange chromatog raphy with a pulsed amperometric detector. Results: Homogeneity of BG6 0 was demonstrated by a single sharp peak in reverse-phase high-perfor mance liquid chromatography, a single band in sodium dodecylsulfate-po lyacrylamide gel electrophoresis, and only one band stained by anti-BG 60 monoclonal antibody. BG60-specific IgE and IgG antibodies wee shown to be present in allergic sera. Six plant lectins were found to react with BG60. On periodate treatment, BG60 reduced binding toward its mo noclonal antibody and human IgE and IgG. Carbohydrate composition anal ysis showed that BG60 contains three kinds of sugars: mannose, N-acety lglucosamine, and fucose (in a ratio of approximately 3:2:1) and a min ute amount of xylose. The carbohydrate content is approximately 7.5%, and peptide content is about 92.5%. Conclusion: A procedure was establ ished for the purification of a large quantity of the BG60 antigen. Th e results suggest that the carbohydrate moiety of antigen BG60 may pla y an important role in the immune response.