Journal: American Journal of Cancer Research

Article Title: Immune response evoked by tumor-associated NADH oxidase (tNOX) confers potential inhibitory effect on lung carcinoma in a mouse model

doi:

Figure Lengend Snippet: The protein expression and immune responses of the human tNOX recombinant proteins in mice. Recombinant human tNOX was expressed in E. coli and purified with His-tag purification resin. A. The expressed, purified and dialyzed proteins were resolved by SDS-PAGE and detected by western blot analysis with anti-His tag and anti-tNOX antibodies, and are shown in Lanes 1, 2 and 3, respectively. The molecular weight (KDa) marker (M) is shown at the left. Arrow indicates the tNOX protein band. B. Blood samples were collected at 0, 14, 28 and 35 days after the first vaccination and antibody titers were measured by ELISA. The specific anti-tNOX antibody response was significantly higher in the tNOX vaccine group compared with unvaccinated controls (*P < 0.05). C. Splenocytes of mice were stimulated with human tNOX protein and IFN-γ-secreting T cells were evaluated by ELISpot assay. Representative images show proliferative spots of splenocytes from mice vaccinated with tNOX (tNOX) or without tNOX (NC). D. The frequencies of tNOX-specific IFN-γ-secreting T cells was significantly higher in the tNOX vaccine group compared to the NC group (*P < 0.05). The average spot counts of the NC and tNOX vaccine groups were 67 ± 13 and 155 ± 42, respectively.

Article Snippet: ELISpot assays were performed using a Mouse IFN-γ ELISpot Development Module (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Recombinant, Purification, SDS Page, Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control