The next day, wells were sequentially incubated with 200 μL blocking buffer (PBS solution, 0.5% Tween 20, 4% dry milk, 10% fetal bovine serum), 100 μL specimen (serum 1 : 50 or stool 1 : 10 in blocking buffer) and 100 μL of horseradish peroxidase goat anti-mouse (Zymed–Invitrogen, San Francisco, CA) immunoglobulin G (IgG) (1 : 4000) or IgA (1 : 2000) in blocking buffer. Incubations were performed for 1 h at room temperature and plates were washed with PBS–Tween 20 (0.05%) between steps. A reaction was developed with 100 μL tetramethylbenzidine substrate (Sigma-Aldrich) for 10 min, stopped with Proteasome inhibitor 100 μL 1 N H2SO4 and the absorbance was determined at a wavelength of 450 nm. All of the specimens were tested
in duplicates and the background reading of noninoculated wells was subtracted Selleckchem Trichostatin A from test wells. Four weeks after the third dose of immunization, animals were challenged with H. pylori. For that, H. pylori SS1 strain (kindly provided by Dr R.M. Peek, Vanderbilt University, Nashville, TN) was grown at 37 °C in brucella broth (Becton Dickinson & Co., Sparks, MD) with 10% fetal bovine serum and antibiotics (vancomycin 10 μg mL−1 and amphotericin B 5 μg mL−1) under microaerophilic conditions (GasPak EZ, Becton Dickinson & Co.) and a suspension of 1–5 × 109 bacteria in PBS administered by gastric gavage every other day for three doses. Four weeks after the challenge, mice were
euthanized and the stomach was harvested to determine the presence of H. pylori organisms. Stomachs were homogenized (Tissue Tearor, Biospec Products, Bartlesville, OK), DNA was extracted (Dneasy Tissue Kit, Qiagen) and subjected to quantitative real-time PCR (Béguéet al., 2006) using primers described previously by Roussel et al. (2007) and targeted to the H. pylori SS1 16S rRNA gene (411–564 bp). Specimens were run in duplicates and positive and negative controls (H. pylori-infected and -uninfected mice, respectively)
were included. In addition, to confirm that the detected signal was due to H. pylori in the specimens, the 16S rRNA gene was amplified (69–611 bp) by regular PCR using primers described by Thoreson et al. (1995), and the resulting amplicon was sequenced at Louisiana State University Health Sciences Center Genomics Core Facility and compared with the H. these pylori SS1 16S rRNA gene (GenBank AY366421). Difference in antibody and H. pylori infection levels between groups were compared using the nonparametric Mann–Whitney U-test (spss 14.0; SPSS, Chicago, IL). The animal experimentation protocol was reviewed, approved and supervised by the Institutional Animal Care and Use Committee of the Research Institute for Children, Children’s Hospital, New Orleans, LA. The results of immunogenicity are shown in Fig. 1. Figure 1a shows serum anti-urease B IgG antibodies. As noted, intranasal administration of rUreB was poorly immunogenic, despite the use of CpG ODN as an adjuvant, and not different from the control group.