Evidence that the serotype b antigenic determinant of Y4 resides in the polysaccharide moiety of lipopolysaccharide. serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to Tegafur immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from serotype d. In conclusion, our results show that the serotype d-specific antigen Tegafur contains the O-antigenic structure of LPS. is a gram-negative, facultatively anaerobic coccobacillus which is associated with aggressively progressing periodontitis. In addition to oral infections, causes serious nonoral infections, such as endocarditis (31). The species is divided into six serotypes (a to f) (8, 26, 39), and fewer than 10% of the strains are nonserotypeable with serological methods (19). Different clonal types exhibit intraspecies differences in virulence characteristics, such as leukotoxin production (5, 39). Tegafur Antibiotic resistance patterns differ among the serotypes and even among the genotypes of the same serotype (20, 22). Although the serotype distribution of seems to differ geographically, evidence from Finland and the United States indicates that serotype b is associated with periodontitis and that serotype c is most frequently found in periodontally healthy individuals (2, 4, 39). The distribution pattern of serotype f is not yet known, whereas serotype e occurs both in periodontally diseased and healthy individuals (4, 6, 12). Among the serotypes, serotype d is infrequent in most populations studied. Thus, its role in the oral flora is even less well known than those of the other serotypes. The serotype-specific antigen of is the immunodominant antigen of the species (27). The composition and location of the antigen have been studied extensively (1, 18, 23, 34). PIK3R5 Researchers agree that the antigen is composed of polysaccharide, but whether it resides in the capsular polysaccharide distinct from lipopolysaccharide (LPS) (1, 3) or in the polysaccharide moiety of LPS (18, 34) is still unclear. Further attempts to characterize the serotype-specific antigens are needed to advance understanding of the role of as a human pathogen and to clarify why serotypes are distributed differently in geographically distant populations, in periodontal health and disease, or among various periodontal diseases. The composition of the LPS of serotype d differs from that of the LPSs of serotypes a, b, c, and e. In contrast to these other serotypes, whose LPS O antigens are composed of repeating disaccharide or trisaccharide units, the O antigen of serotype d is composed of repeating tetrasaccharide units (23). Therefore, in Western blots, the characteristic ladder-like pattern of LPS O antigen is seen in serotype d strains only (14). Furthermore, it was recently shown that eight unique genes code for the serotype d-specific polysaccharide and that the biosynthesis and structure of this polysaccharide antigen are different from those of serotypes b, c, and e (14). However, despite the known structure of the O antigen of serotype d and the unique genes coding for the serotype-specific antigen, it is not known on which surface structure the particular antigen resides. Thus, in the present study we exploited an immunological approach to isolate the serotype d-specific antigen from the outer membrane complex (OMC) by using affinity chromatography. The isolated serotype d-specific antigen was characterized by silver staining, Western blotting, and displacement enzyme-linked immunosorbent assay (ELISA). Additionally, to test the immunogenic properties of the isolated antigen, we immunized rabbits with the antigen and characterized the antibodies. MATERIALS AND METHODS Bacterial strains. The study used five clinical serotype d strains (20) and reference strains ATCC 29523, ATCC 43758, ATCC 33384, IDH781, and IDH1708, representing serotypes a, b, c, d, and e, respectively. The bacterial strains were stored in skim milk at ?70C. Isolation of LPS. LPSs of the strains were isolated as previously described (21). Briefly, the strains were grown on Trypticase soy agar (Difco Laboratories, Detroit, Mich.) plates in 5% CO2 in air at 37C for 2 to 3 3 days. Bacterial colonies were suspended in phosphate-buffered saline (10 mM Tegafur phosphate [pH 7.4], 150 mM NaCl) (PBS), and the cells were broken by sonication for 4.5 min on ice. The total bacterial cell membrane fraction was recovered by centrifugation at 100,000 for 1 h and treated with Sarkosyl (sodium lauryl sarcosinate) (Sigma Chemical Co., St. Louis, Mo.) at room temperature for 1 h, and the perfect solution is was centrifuged at 100,000 for 1 h. The.