Journal: Viruses

Article Title: Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

doi: 10.3390/v16101596

Figure Lengend Snippet: Anti-PoAstV4 spike IgG ELISA: ( A ) ELISA data showing a dose-dependent response towards recombinant PoAstV4 spike antigen in two presumed seropositive (+) pig serum samples. Low-no reactivity was observed in the presumed seronegative (−) pig serum samples from two CDCD piglets. Each sample was measured in triplicate, with the average reported, and error bars represent the standard deviation. ( B ) A matching negative control ELISA using an ELISA plate not coated with an antigen showed low or no reactivity (<0.197 absorbance at 450 nm), supporting the specificity of the ELISA.

Article Snippet: A ninety-six well medium-binding ELISA plate (Corning #9017) was coated with 50 μL of 10 μg/mL purified PoAstV4 spike in phosphate-buffered saline (PBS), PBS alone as a “no antigen” control, covered with microplate sealing tape (Corning #6575) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation, Negative Control

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: ( A ) Illustration of experimental approaches for validation of FLRT3 screening results using 293T-OKT3 cell-based or FLRT3-Fc fusion protein-based systems. ( B to E ) CFSE-labeled human PBMCs from three healthy donors were cocultured with 293T-OKT3 cells transfected with FLRT3, PD-L1, and B7-1 genes followed by analysis of proliferation by CFSE dilution (B and C) and IFN-γ production by ELISA (D and E). Quantification of proliferating CD8 + T cells (B) and IFN-γ production (D) is shown for one representative donor. Each data point represents one technical replicate, and error bars denote SD. (C and E) Percent change mediated by individual genes in CD8 + T cell proliferation (C) and IFN-γ production (E) in comparison to EV control for all three donors in a pairwise fashion. ( F and G ) Human PBMCs from a healthy donor were labeled with CFSE and activated on plates coated with titrated OKT3 and FLRT3-Fc (10 μg/ml). Proliferation of CD8 + T cells was analyzed by CFSE dilution via flow cytometry (F), and IFN-γ production was measured in the supernatants by ELISA (G). Error bars denote SD. n = 3 technical replicates for each data point. Data representative of at least two independent experiments. Statistical significance was determined for (B) and (D) by one-way ANOVA with Tukey’s post hoc test for multiple comparisons and for (C) and (E) by repeated-measures one-way ANOVA with Tukey’s post hoc test for multiple comparisons to account for matched values for individual donors. For all the data, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For ELISA assays evaluating FLRT3 binding to binding partners, 96-well EIA/RIA medium binding ELISA plates (Corning 9017) were coated with appropriate fusion protein (UNC5B-Fc, FLRT3 Fc, LPHN3 Fc, UNC5C Fc, UNC5D Fc, or UNC5A-His) at 5 μg/ml in PBS at 4°C overnight (listed in ).

Techniques: Biomarker Discovery, Labeling, Transfection, Enzyme-linked Immunosorbent Assay, Comparison, Control, Flow Cytometry

Journal: Science Advances

Article Title: The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

doi: 10.1126/sciadv.adj4698

Figure Lengend Snippet: ( A ) Quantification of GFP–NF-κB in UNC5B-Jurkat-NG cells following 16 hours of coculture with EV or FLRT3-293T-OKT3 cells, and NP591 or isotype. Data representative of two experiments. ( B ) Four-day activated human PBMCs from three donors were cocultured with SKOV3 cells with CD3 + CD28 Abs in the presence of NP591 or isotype Ab. Three days later, IFN-γ was measured by ELISA. ( C and D ) PBMCs from three healthy donors (responders) were cocultured with donor PBMCs (stimulators) at a 1:1 ratio for 7 days in the presence of irradiated SKOV3 cells. Isotype control or NP591 was added in the cultures on days 0 and 3. On day 7, supernatants were harvested for IFN-γ (C) and TNF-α (D) analysis by ELISA. Bar graph data points in (A) to (D) represent one replicate, and error bars denote SD. ( E ) FLRT3-293T cells were admixed with human PBMCs and injected in mice intradermally. Mice were treated with NP591 anti–PD-1, NP591 and anti–PD-1 combo, or isotype control intraperitoneally starting on day 7 every 2 to 3 days × 3 weeks, then once per week. n = 12 per group. ( F ) Human PBMCs were injected intravenously followed by intradermal injection of SKOV3 cells 1 day later. Mice were treated with NP591 or isotype control intraperitoneally starting on day 7 every 2 to 3 days × 2 weeks, then once per week. N = 9 for isotype, n = 10 for NP591. Error bars denote SEM in (E) and (F). Data representative of two independent experiments. Statistical significance was determined for (A) by unpaired t test, for (B) by one-way ANOVA with Tukey’s post hoc test for multiple comparisons, for (C) and (D) by two-way ANOVA with Tukey’s post hoc test for multiple comparisons, and for (E) and (F) by paired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For ELISA assays evaluating FLRT3 binding to binding partners, 96-well EIA/RIA medium binding ELISA plates (Corning 9017) were coated with appropriate fusion protein (UNC5B-Fc, FLRT3 Fc, LPHN3 Fc, UNC5C Fc, UNC5D Fc, or UNC5A-His) at 5 μg/ml in PBS at 4°C overnight (listed in ).

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control, Injection

Journal:

Article Title: Isolation and Characterization of Two Proteins from Moraxella catarrhalis That Bear a Common Epitope

doi:

Figure Lengend Snippet: Interaction of purified UspA1 and UspA2 with HEp-2 cells determined by ELISA. HEp-2 cell monolayers cultured in a 96-well plate were incubated with serially diluted UspA1 or UspA2. Bacterial strain O35E was used as the positive control. The dilution of the bacteria was analogous to that of the proteins, beginning with a suspension with an A550 of 1.0. The bound proteins or attached bacteria were detected with a 1:1 pool of antisera to UspA1 and UspA2 as described in Materials and Methods.

Article Snippet: One hundred microliters was added to each well of a 96-well enzyme immunoassay/radioimmunoassay medium binding ELISA plate (Costar Corp., Cambridge, Mass.) and incubated for 16 h at 4°C.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Positive Control