スギ花粉抗原の重合に関する研究

Abstract

In order the immunotherapy of Japanese cedar pollinosis to be more improved, investigations were performed to provide polymerized form of antigen. Preliminary studies have already shown that crude extract was not feasible to be polymerized with glutaraldehyde. It is due to low concentration of protein, and is due to extraordinarily high concentration of pollysaccharides. Investigations were attemped to eliminate polysaccharides from aqueous crude extract by means of ammonium sulfate precipitation and DEAE, CM cellulose chromatography, according to the method by YASUEDA et al with slight modifications.In its procedure finally yielding Sugi Basic Protein (SBP), one of the by-products, Fraction b (Fr.b) was proved to be polymerized with glutaraldehyde because of its highly purified form of protein and because of extensive exclusion of polysaccharides (protein: polysaccharide_??_1:1). Approximately 50mgs of native Fr.b were obtained from 50 grams of original pollen, the amount practically useful for clinical application. Examination by Sephadex G-200gel chromatography revealed native Fr.b had moderately been polymerized to 500, 000 to 600, 000 in molecular weight. Reduction of allergenicity of polymerized Fr.b was estimated using ELISA direct titration method and inhibition test, and evidence was obtained that IgE antibody binding activity of polymerized Fr. b had been reduced approximately one-fourth of its original material (native Fr. b). It was also revealed by ELISA that 1mg's protein of native Fr.b had a content of 0.34 to 0.43mgs of SBP. Polymerized Fr.b showed 10-1to10-4fold reactivity by human intracutaneous test when compared with native Fr.b, and 2-1to2-2fold by P-K endpoint titration. Polymerized Fr.b was as immunogenic as, or a little more immunogenic than native Fr.b by intraperitoneal immunization against BALB/c mice.In coclusion, Japanese cedar pollen antigen was proved to be polymerized after extensive elimination of polysaccharides. The results obtained strongly suggest that polymerized Fr.b has reduced allergenicity while retaining immunogenicity. Evidences described are closely related to the results with polymerized mite antigen previously reported by KOSEKI. Further clinical investigations will be warranted by the use of polymerized Fr.b.