Feces were collected, weighed and re-suspended in PBS containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (Boehringer Mannheim Co., USA) and 1% Selleck ROCK inhibitor Bovine Serum Albumin (BSA) (Fisher Scientific
Co., USA) at a ratio of 1 g feces per 5 mL inhibitory solution. After 15 min on ice, the samples were shaken and then centrifuged at 22,000 × g for 10 min, and the supernatants were stored at −80 °C until use. Total immunoglobulin G (IgG) and A (IgA) isotypes and the IgG1 or IgG2a antibody subclasses specific for BfpA and intimin were evaluated by ELISA. Briefly, microtiter plates were coated overnight at 4 °C with 5 μg/mL recombinant BfpA or intimin (purified in our laboratory) in 100 μL PBS. The plates were then blocked with 10% BSA in PBS for 1 h at room temperature. After each incubation, the plates were washed three times with PBS containing 0.05% Tween-20 (PBST). Aliquots of serum and fecal extracts were added to individual wells (100 μL), and the plates were incubated for 1 h at room temperature. After washing, the plates were incubated with 100 μL peroxidase-conjugated goat anti-mouse IgG or anti-mouse IgA or anti-mouse IgG1 and IgG2a (Southern Biotechnologies, USA) at a dilution of 1:1000 in the same diluent pursued by 1 h incubation at room temperature. The peroxidase activity was measured using the o-phenylenediamine (OPD) substrate and
read at a wavelength Anti-diabetic Compound Library of 450 nm. Spleens were recovered from immunized mice (5 animals per group) 15 days after the final immunization. Cell Histamine H2 receptor suspensions were prepared at a concentration of 5 × 106 cells/mL in RPMI medium (Gibco, USA) containing polymixin (1 μg/mL) and were plated in 24-well plates. Cells were left unstimulated or were stimulated for 48 h with extracts of Smeg, BCG, purified BfpA, purified intimin or ConA (Sigma, USA) at a concentration of 5 μg/mL at 37 °C in 5% CO2. Cytokine secretion was evaluated using the Cytometric Bead Array Th1/Th2 Kit (CBA; BD Bioscience, USA) and samples were read on a FACS Calibur flow cytometer (BD Biosciences, USA). Each experiment was repeated three
times. To evaluate the ability of the anti-recombinant BfpA and intimin antibodies to interfere with the adhesion of EPEC to host cells, a standard assay using HEp-2 target cells was used. Hep-2 cells were maintained in DMEM supplemented with 10% SFB in a humidified atmosphere containing 5% CO2 at 37 °C. To evaluate the inhibitory action of the specific antibodies, serum or fecal samples were incubated at a ratio of 1:4 with 107 EPEC bacteria for 1 h at 37 °C before being added to the Hep-2 cultures. After the incorporation of the bacteria, the HEp-2 monolayers were kept at 37 °C for 3 h. The HEp-2 monolayers were washed with PBS, fixed with methanol and stained with Giemsa solution to visualize the adherent bacteria by light microscopy.