Journal: International Journal of Molecular Sciences
Article Title: Comparison of the L3-23K and L5-Fiber Regions for Arming the Oncolytic Adenovirus Ad5-Delta-24-RGD with Reporter and Therapeutic Transgenes
doi: 10.3390/ijms26083700
Figure Lengend Snippet: Characterization of Ad5Δ24RGD expressing the wild-type murine PD-1, wild-type human PD-1 or human mutant high-affinity PD-1 HA ectodomain. ( A ) Evaluation of the hPD-1 and hPD-1 HA ectodomain concentrations in the supernatants of human and mouse cells by an enzyme-linked immunosorbent assay (ELISA). The cells (1 × 10 5 /well) were infected in suspension at an MOI of 5 IFU/cell for A549, 50 IFU/cell for LN18 and CT-2A, and 100 IFU/cell for GL261. The supernatants were collected at 48 hpi. The data are shown as means (SD) of two independently repeated experiments ( N = 2). ( B ) Comparative analysis of the competitive antagonist activity of the hPD-1 and hPD-1 HA ectodomains. At seventy-two hours after the infection of 5 × 10 6 A549 cells with OAds at an MOI of 20 IFU/cell, the conditioned medium was collected, concentrated 20-fold, and analyzed using a PD-1-biotin/PD-L1 ELISA. The recombinant human PD-1 protein (active) (ab174035, Abcam) was used as a control. The data are shown as means (SD) of two independently repeated experiments ( N = 2). ( C ) A comparative analysis of the total virus yields was performed by harvesting the cell lysates along with the supernatants at 24, 48, 72, and 120 hpi. A549 cells (1 × 10 5 ) were infected in suspension with the indicated OAds at an MOI of 10 IFU/cell. The titer is reported in infectious units per milliliter (IFU/mL) and was determined using immunocytochemistry with anti-capsid staining. The data are shown as means (SD) of three independently repeated experiments ( N = 3). ( D ) Comparison of the IC 50 values obtained from viability curves after infection of human cells (5 × 10 3 /well) and mouse cells (2.5 × 10 3 /well) in suspension with serial 3-fold dilutions of the indicated OAds starting from an MOI of 333 IFU/cell for A549 cells, 1000 IFU/cell for LN18 cells, 2000 IFU/cell for GL261, and 666 IFU/cell for CT-2A cells. The data were collected on Day 5 postinfection. Two (human cells) or three (mouse cells) independently repeated experiments with technical triplicates were performed. The data are shown as means (SD) of independently repeated experiments ( N = 2 − 3). Only ≥2-fold differences in the mean IC 50 values are indicated. ( E ) Comparison of the sizes of MTT-stained plaques in the cancer cell monolayers under the agarose overlay. The cells were seeded in 6-well plates (6 × 10 5 cells/well for A549 and 9 × 10 5 for LN18) and infected the following day with appropriate dilutions of viruses determined empirically. The sizes of 40–70 random plaques from two to three wells were taken for analysis from each independent experiment. The total number ( n ) of analyzed plaques collected from two independently repeated experiments ( N = 2) are indicated for each group. The data are shown as means (SD) of the total number ( n ) of analyzed plaques. Only ≥1.3-fold differences in the mean plaque sizes are indicated.
Article Snippet: The concentrated supernatants were analyzed using the PD-1 [Biotinylated]:PD-L1 inhibitor screening ELISA kit (#EP-101-96tests, ACROBiosystems, Newark, DE, USA).
Techniques: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Suspension, Activity Assay, Recombinant, Control, Virus, Immunocytochemistry, Staining, Comparison
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