1997. from bacterial isolation (4). A breakthrough for this type of vaccines was made in 2004 by Verez-Bencomo et al. (33) when they reported the large-scale synthesis and the introduction of a synthetic oligosaccharide vaccine for type b for human in Cuba. Pneumococcal disease is a major NB-598 Maleate public health problem worldwide, and it is estimated that 1.6 million people die from this infection each year, 1 million of whom are children (36). Capsular polysaccharides (PS) are well known as the major virulence factors of type 14 PS (Pn14PS) consists of biosynthetic repeating units of the tetrasaccharide (19) {6)-[-d-GalR595 lipopolysaccharide; Ribi ImmunoChem Research, Inc., Hamilton, MT] and 20 g of Quil-A [Superfos Biosector, Vedbaek, Denmark] per animal) and injected intracutaneously at the four sites, as mentioned above. A booster of 2.5 g of carbohydrate was given on day 35 without adjuvant. Using a retro-orbital puncture, blood samples were taken by from isofluran-anesthetized mice 1 week before the booster and 2 and 3 weeks after the booster. Measurement of Pn14PS-, protein carrier-, and spacer-specific antibodies by ELISA. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the anti-Pn14PS antibodies, as described previously (4, 18). Briefly, diluted sera were incubated for 1 h at 37C in flat-bottom plates (Corning, Inc., Corning, NY) which were coated with Pn14PS (0.3 g/well) and blocked with 3% gelatin. After a washing step, horseradish peroxidase-conjugated goat anti-mouse IgM or IgG (Nordic Immunology Laboratories, Tilburg, The Netherlands) was incubated for 1 h at 37C. A mixture of 3,3,5,5-tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO), and H2O2 (Sigma Chemical Co.) was then added to visualize the amount of bound peroxidase. The reaction was stopped with the addition of 0.5 M H2SO4. Optical density (OD) values were obtained with a microtiter plate spectrophotometer at 450 nm (Bio-Rad, model 3550 UV; Bio-Rad Laboratories, Hercules, CA). NB-598 Maleate Antibody titers were expressed as the log10 of the dilution giving twice the OD obtained for control mice (immunized with saline) with a cutoff value of 0.2. CRM197- and BSA-mannose-coated plates (0.1 g/well) were also used to measure anti-protein carrier and anti-spacer titers, respectively. BSA-mannose was constructed by coupling 6-aminohexyl -d-mannopyranoside, via diethyl squarate, to BSA. Detection of oligosaccharide-specific antibodies. In order to investigate the immune response Rabbit polyclonal to ABCA6 to the oligosaccharide fragments that did not induce anti-Pn14PS antibodies, ELISAs were performed after preincubating the sera with BSA-mannose to block the antibodies recognizing the spacer molecule (-C6H12-NH-C4O2-NH-). After the uncoated plates were blocked with 2% gelatin in phosphate-buffered saline (PBS) and washed with NB-598 Maleate PBS-0.05% Tween 20 several times, diluted sera (1:100 in PBS supplemented with 0.05% Tween 20 and 3% Protifar) were incubated with BSA-mannose in a concentration ranging from 0 to 100 g/ml for 1 h at 37C and left overnight at 4C. The mixtures were then transferred to three differently coated plates (0.1 g/well): BSA-mannose, BSA-conjugates corresponding with CRM197 conjugates, and BSA-DM66 (for the structures, see Table ?Table1).1). NB-598 Maleate The amount of specific antibodies in these absorbed sera was detected by ELISA method as described above. The results were expressed as the OD changes of the sera incubated with BSA-mannose compared to sera alone. Measurement of avidity. The antibody avidity of mouse sera that recognized Pn14PS as the coating material was measured by ELISA using chaotropic sodium thiocyanate (NaSCN; Sigma Chemical Co.), as previously described (18, 25). Briefly, Pn14PS-coated plates were incubated with diluted sera (1:25 in PBS, 0.05% Tween 20 and 3% Protifar) and washed. Series of NaSCN concentrations (0 to 3.0 M) were incubated on the plates at 37C for 15 min. After five washes, horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000) in PBS was added, and the solution was incubated for 1 h at 37C. The amount of antibodies left was detected by ELISA as described above. In the present study, the avidity index (AI) is expressed as the concentration of NaSCN needed to reduce the OD450 by 50%. The absorbance value of sera without NaSCN should be at least 0.5. Measurement of phagocytosis titer..