Sera were diluted 1640,000 before use. ELISA. **, 0.05.(DOC) pone.0047219.s002.doc (24K) GUID:?37CB89EC-46DC-4EE0-82DC-BF58227A0BE4 Physique S3: Intracellular cytokine staining of CD4+ T cells. Mice were immunized with HCA587+CpG+ISCOM,CpG+ISCOM, or PBS, and boosted 21 days later. Splenocytes were harvested Ethoxyquin 14 days after the boost and stimulated with HCA587 protein for 24 h. Brefeldin A (10 g/ml) was added 18 hours before harvesting the cells from the culture. Intracellular staining was performed for IFN- and IL-10 by gating on CD4+ T cells.(DOC) pone.0047219.s003.doc Ethoxyquin (88K) GUID:?A970CFD5-78AE-413A-8CE2-FEF301DE6EBC Physique S4: Detection of IgG subclass of anti-HCA587 antibodies in the serum of HCA587 protein vaccine immunized mice. C57BL/6 mice (n?=?9) were immunized with HCA587 protein vaccine twice at a 3-week interval. Sera were harvested 14 days after the boost. Levels of HCA587-specific IgG1 and IgG2c were measured by ELISA. Sera were diluted 1640,000 before use. **, strain BL21 (DE3). The fusion protein was first affinity-captured using Glutathione Sepharose 4B (GE Healthcare). After removal of the GST-tag by cleavage with PPase, the recombinant HCA587 protein was further purified by ion-exchange chromatography (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 200, GE Healthcare). The purity of recombinant HCA587 protein were analyzed using reverse phase-high performance liquid chromatography (RP-HPLC) and the full sequence of recombinant HCA587 protein was verified by tandem MS. The purity of HCA587 protein used for vaccine was more than 95%, with endotoxin at a level below 3.1 units/100 g protein. All-phosphorothioate modified CpG oligonucleotide (CpG ODN) 1826 (5 CTCCATGACGTTCCTGACGTT-3) was synthesized by the Shanghai Sangon Biological Engineering & Technology and Support (Shanghai, China). Immune-stimulating complex (ISCOM) AbISCO 100 was purchased from Corporation Isconova AB, Sweden. Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA) were purchased from Sigma-Aldrich (St. Louis, MO). Immunization Mice were immunized s.c. at the base of the tail with 10 g of HCA587 protein. Different adjuvants or combinations of them were tested for their ability to induce optimal cellular and/or humoral responses, including CFA (50 l), CpG ODN (12 g), ISCOM (12 g), or CpG ODN plus ISCOM. A total volume of 100 l was administrated using PBS as a vehicle. Each animal received two injections at a 3 week interval. For the CFA group, IFA was used for the second immunization. Controls were set up by immunizing age-matched mice with PBS, HCA587 protein alone, or adjuvants alone. Antibody ELISA Assay Serum samples were collected from the mice at different time points. HCA587-specific antibody titers were detected using enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA plates were coated with HCA587 protein at 1 g/ml in PBS, and then blocked with 2% bovine serum albumin. Serially diluted mouse serum samples were added into each well and incubated at 37C for 2 hours. PTGIS Mouse IgG Ethoxyquin bound around the plates was detected by horseradish peroxidase-conjugated goat anti-mouse IgG (Promega, Madison, WI) using tetramethylbenzidine (TMB; Tiangen Biotechnology Corporation, Beijing, China) as a peroxidase substrate. The reaction was stopped by the addition of 2 M H2SO4, and the absorbance was read at 450 nm. To detect mouse serum IgG subclass, biotin-labeled anti-IgG1 and anti-IgG2c antibodies (Bethyl, Montgomery, TX) and streptavidin-conjugated HRP (BD, San Jose, CA) were used. IFN- ELISPOT Assay After lysing RBCs, splenocytes were resuspended at a density of 5106/ml, and 100 l of this suspension was then incubated with HCA587 protein (2.5 g/ml) or ovalbumin (OVA; 2.5 g/ml, Sigma-aldrich) in ELISPOT plates coated with anti-IFN- capture Abs (Mabtech, Nacka, Sweden). After incubation for 20 hours at 37C, cells were removed, and the plates were developed with a biotinylated anti-mouse IFN- detecting Abs and streptavidin-alkaline phosphatase (Mabtech). The Ethoxyquin dark violet spots displayed around the plate membranes were automatically counted with the ELISPOT reader (Sage Creation, Beijing, China). Cytokine Assay RBC-depleted splenocytes (5106/ml) from vaccinated mice were prepared and stimulated with HCA587 protein (10 g/ml) or ovalbumin (10 g/ml) for 24 hours at 37C. The supernatants were collected and analyzed for IL-4, IL-5, IFN-, IL-2, TNF-, IL-10, and IL-17 with commercial available ELISA kits (Dakewe Biotechnology, Beijing, China) according to the manufactures procedures. Comparable procedures were followed to measure the levels of IFN-, IL-12, IL-6, IL-10, and TNF- in the serum of immunized mice. For intracellular cytokine staining, brefeldin A (10 g/ml; Biolegend) was added 18 hours before harvesting the cells from the culture. The cells were first surface stained for CD4 and CD8, then fixed and permeabilized (Biolegend) according to the manufacturers instruction,.