gingivalis was inserted into the p-MAL plasmid pMD157, followed by transfection to E. coli and incubation. After 1 or 2 days of incubation, the E. coli suspension was centrifuged and the pellet was homogenized. The homogenized suspension

was subjected to the dialysis treatment, gel-filtration chromatography, and ion-exchange chromatography. Finally, isolation DMXAA chemical structure of the antigen was performed using amylose resin column affinity chromatography, and 25k-hagA was obtained via cleavage treatment of 25k-hagA-MBP using Factor Xa (New England BioLabs, Ipswich, MA). For sublingual immunization on days 0, 7, and 14, mice were anesthetized with pentobarbital, and 30 μL of phosphate-buffered saline (PBS) containing 50 μg of 25k-hagA-MBP was delivered with a micropipette applied against the ventral side of the tongue while directed toward the floor of the mouth. Mice were immunized with 7.5 μL of antigen four times (total volume = 30 μL). Ten minutes of interval were set between each administrations. A nonimmunized

group was PBS treated. Animals were maintained with their heads placed in ante flexion for 30 s during each delivery. Serum and saliva were collected from each group to examine the 25k-hagA-MBP-specific Ab responses. Ab titers were detected using an enzyme-linked immunosorbent assay (ELISA) as described previously (Maeba et al., 2005). Briefly, plates were coated with 25k-hagA-MBP (5 μg mL−1). After Selleck PD98059 washing with PBS containing 0.05% Tween 20, plates were blocked with PBS containing 1% bovine serum albumin. Next, serial dilutions of serum or saliva samples were added in duplicate. The starting dilution of the serum was 1 : 26, while that of the saliva was 1 : 22. The plates were incubated for 5 h at room temperature, washed, and then incubated with horseradish peroxidase-labeled goat anti-mouse heavy chain γ, γ1, γ2a, γ2b, γ3, or α-specific antibodies (Southern Biotechnology Associates, Birmingham,

AL) at 4 °C for 20 h. Finally, 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic Lck acid) (ABTS) with H2O2 (Moss, Inc., Pasadena, MD) was added for color development. Endpoint titers were expressed as the reciprocal log2 of the last dilution, which gave an optical density at 415 nm of 0.1 greater than that of nonimmunized control samples after 15 min of incubation. Single-cell suspensions were obtained from the salivary gland 7 days after the last immunization. Briefly, salivary glands were carefully extracted, teased apart, and dissociated using 0.3 mg mL−1 collagenase (Nitta Gelatin Co. Ltd, Osaka, Japan) in RPMI-1640 (Wako Pure Chemical Industries Ltd, Osaka Japan). Mononuclear cells were obtained at the interface of the 50% and 75% layers of a discontinuous Percoll gradient (GE Healthcare UK, Ltd, Little Chalfont, Buckinghamshire, UK) (Maeba et al., 2005). To assess the numbers of antigen-specific AFCs, an enzyme-linked immunospot (ELISPOT) assay was performed as described previously (Yamamoto et al., 1997).