Each serum sample is shown as S1, S2, S3, S4, S5 and S6. antibodies were depleted from Ginsenoside Rh1 immune sera in order to preferentially study IgG antibodies, the proportion of young adult sera showing more than 60% inhibition in opsonic capacity by 10 mM of L-rhamnose increased from 33% (11/31) to 68% (21/31). On the other hand, IgM depletion did not alter the proportion for old adult sera. Therefore, young and old adults may produce different antigen binding profiles of IgG antibodies against serotype 23F PS. Introduction is a significant pathogenic bacterium that causes several diseases such as pneumonia, bacteremia, meningitis, and otitis media [1]. Among many kinds of surface molecules on pneumococci, capsular polysaccharide (PS) is one of the major virulence factors [2]. Pneumococcal capsular PS is a polymer of carbohydrate repeating units, and so far at least 93 different serotypes are described based on the structure of PS [3]C[6]. Since antibodies against pneumococcal PS are highly protective, adults are immunized with a 23-valent pneumococcal PS vaccine (PPV23) and young children are immunized with several pneumococcal conjugate vaccines (PCVs), which are produced Gpc3 by conjugating 7C13 different capsular PS to a carrier protein [7]C[11]. With increasing numbers of conjugates in a vaccine, the complexity of a vaccine greatly increases, and thus there is a need to search for a simple epitope that can elicit antibodies against pneumococci [12]C[15]. Serotype 23F capsular PS has tetrasaccharide repeating units containing one glucose, Ginsenoside Rh1 one galactose and two L-rhamnose residues (Figure 1). One of the two rhamnose residues forms a branch of the backbone (Figure 1) [16], [17] and previous studies showed that the structures including branched L-rhamnose Ginsenoside Rh1 is the dominant epitope recognized by horse and rabbit antisera [16], [18]. However, it has not yet been determined whether antibodies targeting rhamnose are functional (i.e., opsonic). If functional, it is unclear whether rhamnose-specific antibodies remain functional among old adults, inasmuch as antibodies from old adults tend to be poorly functional [16], [18]. Open in a separate window Figure 1 Structure of the PS of serotype 23F. In this study, therefore, we used an opsonophagocytosis assay to determine if L-rhamnose-containing structure is a functional epitope of human antibodies specific for pneumococcal serotype 23F PS from a large number of serum samples obtained from young or old adults immunized with PPV23. We studied these rhamnose specific antibodies to investigate the functional difference in the anti-23F antibody repertoire between young and old adults. Materials and Methods Ginsenoside Rh1 Serum samples Two groups of anonymous human sera were used. One group was sera from old adults (70C79 years of age; N?=?44), who had received one PPV23 at least 5 years prior to enrollment. They were immunized with 0.5 ml of pneumococcal PS vaccine (PPV23) (Pneumovax?, Ginsenoside Rh1 Merck, Whitehouse Station NJ) one month before phlebotomy [19]. Pre-immune sera were obtained before enrollment vaccination, and analyzed for functional antibody activity in this study. Old adult serum samples used in this study were described previously [19]. The other group (N?=?55) was from young adults (<42 years of age) who were immunized with PPV23 one month before phlebotomy. Young adult serum samples were obtained from M. Blake (Bethesda, MD) and described in our previous study [20]. Inhibition ELISA Inhibition ELISA was performed as described in 3rd generation pneumococcal antibody ELISA (www.vaccine.uab.edu) with some modifications. Wells of medium-binding microtiter plates were coated at 37C with a pre-determined concentration (10 g/ml) of capsular PS serotype 23F (ATCC, Rockville, MD) for 5 hr in phosphate buffered saline (PBS) with 0.02% NaN3. Serum samples (diluted 1200) were pre-absorbed.