solid phase sandwich elisa  (R&D Systems)

Journal: Gene Therapy

Article Title: The co-delivery of Programmed Death 1 ligands enhances and prolongs rAAV-mediated gene expression in pre-immunized mice

doi: 10.1038/s41434-025-00588-9

Figure Lengend Snippet: A Western blot showing PD-L1 (top) and VEGF-B186 (middle) produced in cell lysates and media of transfected 293T cells, respectively, with (+) and without (–) codon-optimization. B BaF3-R1 cell viability after incubation with VEGF-B186-containing media of PD-L1-VEGF-B186 and VEGF-B186-PD-L1-transfected 293T cells. Data are presented as mean (dots) (SD) of two technical replicates from a representative experiment. C Western blot showing PD-L2 (top) and VEGF-B186 (middle) produced in cell lysates and media of transfected 293T cells, respectively. D BaF3-R1 cell viability after incubation with VEGF-B186-containing media of VEGF-B186-PD-L2-transfected 293T cells. Data are presented as mean (dot) (SD) of two technical replicates from a representative experiment. E Western blot showing muSEAP (top) produced into the media and PD-L1 (second row) and PD-L2 (third row) in the cell lysates of transfected 293T cells. F Luminescence assay measuring muSEAP secretion into the media of transfected 293T cells. Data are presented as individual values (dot) and mean (column) (SD) of two independent experiments. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis. Mock-treated cells and media were used as negative controls. G PD-L1 and PD-L2 detection after pull-down with PD-1-His beads (+). Non-coated beads were used as a control (−). H . Luminescence assay measuring muSEAP secretion into the media of rAAV6 transduced 293T cells. Data are presented as individual values (dots) and mean (column) (SD) of two independent experiments. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis. Mock-treated cells and media were used as negative controls, and GAPDH was used as a housekeeping control. MW molecular weight, GAPDH glyceraldehyde 3-phosphate dehydrogenase, RLU relative light unit.

Article Snippet: Primary antibodies used were human PD-L1 antibody (cat# 13684, Cell Signaling Technologies, Danvers, MA, USA), human PD-L2 antibody (cat# 82723, Cell Signaling Technologies), human VEGF-B167/186 antibody (cat# AF751, R&D Systems, Minneapolis, MN, USA), human alkaline phosphatase antibody (cat# sc-398461, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (cat# 2118, Cell Signaling Technologies).

Techniques: Western Blot, Produced, Transfection, Incubation, Luminescence Assay, Control, Molecular Weight

Journal: Gene Therapy

Article Title: The co-delivery of Programmed Death 1 ligands enhances and prolongs rAAV-mediated gene expression in pre-immunized mice

doi: 10.1038/s41434-025-00588-9

Figure Lengend Snippet: A A schematic overview of the 5 WPGT (top) and 12 WPGT (bottom) mouse study. Weeks when blood samples were taken are indicated in blue. Figure generated with BioRender. muSEAP measured from the PI mice serum in the 5 WPGT study ( B ) and the 12 WPGT study ( C ), and in naïve mice serum in the 5 WPGT study ( D ) and the 12 WPGT study ( E ). Multiple linear regression was used for statistical analysis. Significances are shown as the effect of treatment as a function of time. Data are presented as means of two independent experiments with LOWESS smoothing and 95% confidence intervals. Mice treated with PBS were used as negative controls. The number of samples are as follows: 5 WPGT study PI mice, rAAV6-muSEAP-PD-L1 n = 6, rAAV6-muSEAP-PD-L2 n = 4, rAAV6-muSEAP n = 4, negative control n = 2; 5 WPGT study naïve mice, rAAV6-muSEAP-PD-L1 n = 4, rAAV6-muSEAP-PD-L2 n = 4, rAAV6-muSEAP n = 4, negative control n = 2, 12 WPGT study PI mice, rAAV6-muSEAP-PD-L1 n = 6, rAAV6-muSEAP-PD-L2 n = 6, rAAV6-muSEAP n = 6, negative control n = 2, 12 WPGT study naïve mice, rAAV6-muSEAP-PD-L1 n = 6, rAAV6-muSEAP-PD-L2 n = 6, rAAV6-muSEAP n = 6, negative control n = 2. WPGT weeks post gene transfer, RLU relative light unit.

Article Snippet: Primary antibodies used were human PD-L1 antibody (cat# 13684, Cell Signaling Technologies, Danvers, MA, USA), human PD-L2 antibody (cat# 82723, Cell Signaling Technologies), human VEGF-B167/186 antibody (cat# AF751, R&D Systems, Minneapolis, MN, USA), human alkaline phosphatase antibody (cat# sc-398461, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (cat# 2118, Cell Signaling Technologies).

Techniques: Generated, Negative Control

Journal: Gene Therapy

Article Title: The co-delivery of Programmed Death 1 ligands enhances and prolongs rAAV-mediated gene expression in pre-immunized mice

doi: 10.1038/s41434-025-00588-9

Figure Lengend Snippet: A PD-L1. B PD-L2. C muSEAP. Gene expression was normalized against a housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) and is shown as a fold change. Data are presented as individual values (dot = 5 WPGT study, square = 12 WPGT study), with different RNA extraction sites indicated with solid or open symbols, and as median (columns) (IQR) from a representative experiment of three experiments. Mice treated with PBS were used as a negative control. The Mann-Whitney test was used for statistical analysis. The number of samples are as follows: 5 WPGT, PI rAAV6-muSEAP-PD-L1 n = 5, PI rAAV6-muSEAP-PD-L2 n = 3, PI rAAV6-muSEAP n = 3, PI negative control n = 1, naïve rAAV6-muSEAP-PD-L1 n = 3, naïve rAAV6-muSEAP-PD-L2 n = 3, rAAV6-naïve muSEAP n = 3, naïve negative control n = 1; 12 WPGT PI rAAV6-muSEAP-PD-L1 n = 6, PI rAAV6-muSEAP-PD-L2 n = 6, PI rAAV6-muSEAP n = 6, PI negative control n = 2, naïve rAAV6-muSEAP-PD-L1 n = 6, naïve rAAV6-muSEAP-PD-L2 n = 6, naïve rAAV6-muSEAP n = 6, naïve negative control n = 2. WPGT weeks post gene transfer.

Article Snippet: Primary antibodies used were human PD-L1 antibody (cat# 13684, Cell Signaling Technologies, Danvers, MA, USA), human PD-L2 antibody (cat# 82723, Cell Signaling Technologies), human VEGF-B167/186 antibody (cat# AF751, R&D Systems, Minneapolis, MN, USA), human alkaline phosphatase antibody (cat# sc-398461, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (cat# 2118, Cell Signaling Technologies).

Techniques: Gene Expression, RNA Extraction, Negative Control, MANN-WHITNEY

Journal: Gene Therapy

Article Title: The co-delivery of Programmed Death 1 ligands enhances and prolongs rAAV-mediated gene expression in pre-immunized mice

doi: 10.1038/s41434-025-00588-9

Figure Lengend Snippet: The prevalence of CD3- ( A ), CD4- ( B ), and CD8-positive cells ( C ), as well as cells expressing Ki67 proliferation marker ( D ) and immune checkpoint molecule PD-1 ( E ), is shown as the stained cell area in the tissue. Data are shown as individual values (dot = 5 WPGT, square = 12 WPGT), and medians are shown as columns (IQR). The Kruskal-Wallis test with Dunn’s post-hoc test was used for statistical analysis between the treatment groups within PI and naïve groups, and the Wilcoxon test was used for statistical analysis between the corresponding PI and naïve groups. The number of samples are as follows: 5 WPGT study, PI rAAV6-muSEAP-PD-L1 n = 4, PI rAAV-muSEAP-PD-L2 n = 4, PI rAAV6-muSEAP n = 4, PI negative control n = 2, naïve rAAV6-muSEAP-PD-L1 n = 4, naïve rAAV6-muSEAP-PD-L2 n = 4, naïve rAAV6-muSEAP n = 4, naïve negative control n = 2; 12 WPGT study, PI rAAV6-muSEAP-PD-L1 n = 6, PI rAAV-muSEAP-PD-L2 n = 6, PI rAAV6-muSEAP n = 6, PI negative control n = 2, naïve rAAV6-muSEAP-PD-L1 n = 6, naïve rAAV6-muSEAP-PD-L2 n = 6, naïve rAAV6-muSEAP n = 6, naïve negative control n = 2. WPGT weeks post gene transfer.

Article Snippet: Primary antibodies used were human PD-L1 antibody (cat# 13684, Cell Signaling Technologies, Danvers, MA, USA), human PD-L2 antibody (cat# 82723, Cell Signaling Technologies), human VEGF-B167/186 antibody (cat# AF751, R&D Systems, Minneapolis, MN, USA), human alkaline phosphatase antibody (cat# sc-398461, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (cat# 2118, Cell Signaling Technologies).

Techniques: Expressing, Marker, Staining, Negative Control

Journal: Gene Therapy

Article Title: The co-delivery of Programmed Death 1 ligands enhances and prolongs rAAV-mediated gene expression in pre-immunized mice

doi: 10.1038/s41434-025-00588-9

Figure Lengend Snippet: AAV6 antibodies in PI mice in the 5 WPGT study ( A ) and 12 WPGT study ( B ), and in naïve mice in the 5 WPGT study ( C ) and 12 WPGT study ( D ). Data are presented as median (dots) (range) from representative experiments of two (5 WPGT study) and one (12 WPGT study) experiments. The Kruskal-Wallis test with Dunn’s post hoc test was used for statistical analysis. rAAV6-muSEAP-PD-L1, rAAV6-muSEAP-PD-L2, and rAAV6-muSEAP termination serum samples were used in both studies. Mice treated with PBS were used as negative controls. The number of samples are as follows: 5 WPGT study PI mice, before pre-immunization n = 2, before GT n = 2, rAAV6-muSEAP-PD-L1 n = 3, rAAV6-muSEAP-PD-L2 n = 3, rAAV6-muSEAP n = 3, negative control n = 1; 5 WPGT study naïve mice, before GT n = 2, rAAV6-muSEAP-PD-L1 n = 3, rAAV6-muSEAP-PD-L2 n = 3, rAAV6-muSEAP n = 3, negative control n = 1; 12 WPGT study PI mice, before pre-immunization n = 2, before GT n = 3, rAAV6-muSEAP-PD-L1 n = 3, rAAV6-muSEAP-PD-L2 n = 3, rAAV6-muSEAP n = 3, negative control n = 1; 12 WPGT study naïve mice, Before GT n = 3, rAAV6-muSEAP-PD-L1 n = 3, rAAV6-muSEAP-PD-L2 n = 3, rAAV6-muSEAP n = 3, negative control n = 1. PD-L1 antibodies in mouse serum of naïve mice from the 5 WPGT study ( E ) and 12 WPGT study ( F ), and PI mice from the 5 WPGT study ( G ) and the 12 WPGT study ( H ). rAAV6-muSEAP-PD-L2 (Negative control 1) and PBS (Negative control 2) treated mice were used as negative controls. Data are representative of two independent experiments. GT gene transfer, WPGT weeks post gene transfer.

Article Snippet: Primary antibodies used were human PD-L1 antibody (cat# 13684, Cell Signaling Technologies, Danvers, MA, USA), human PD-L2 antibody (cat# 82723, Cell Signaling Technologies), human VEGF-B167/186 antibody (cat# AF751, R&D Systems, Minneapolis, MN, USA), human alkaline phosphatase antibody (cat# sc-398461, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (cat# 2118, Cell Signaling Technologies).

Techniques: Negative Control