elisa well plates medium binding  (Greiner Bio)

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: NPJ Regenerative Medicine

Article Title: A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins

doi: 10.1038/s41536-023-00297-0

Figure Lengend Snippet: a Sequence motif generated from PlGF 123-138 used with ScanProsite against the human UniProtKB database to find ECM-binding protein candidates. The 6 lysine and arginine blocks (1-6) are represented in red. The 5 blocks without lysines and arginines are represented in grey. b Aligned motif from ECM-binding protein candidates listed in alphabetic order. Blocks of lysine and arginine are in red. c Pairwise sequence alignments between PlGF 123-138 and the motif from protein candidates using the Needleman-Wunsch algorithm. Two dots indicate the same amino acids and one dot indicates amino acids with similar proprieties. Needleman–Wunsch scores (N-W) are shown. d Binding of the protein candidates to ECM proteins. ELISA plates were coated with the protein candidates or bovine serum albumin (BSA) control and incubated with ECM proteins. Bound ECM proteins were detected with antibodies. Graphs show fold changes in binding over BSA control. n = 4. e Binding affinity of PlGF, AREG, and NRTN to ECM proteins. ELISA plates were coated with ECM proteins and incubated with PlGF, AREG, or NRTN at increasing concentrations. Bound PlGF, AREG, and NRTN were detected with antibodies. Graphs show the K D obtained from the binding curves. n = 3. For ( d , e ) data are means ± SEM. For ( d ) one-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. For ( e ) two-tailed Student’s t test. *** P < 0.001, otherwise indicated. n.s., non-significant.

Article Snippet: ELISA plates (96-Well Medium Binding, Greiner bio-one) were coated for 1 h at 37 °C with 100 nM solutions of PlGF-2, AREG, NRTN, CXCL-9, CXCL-12 (SDF-1β), CXCL-17, VEGF-A, or BSA (Sigma, A8806).

Techniques: Sequencing, Generated, Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Incubation, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins

doi: 10.1038/s41536-023-00297-0

Figure Lengend Snippet: a AREG fragment sequences fused to GST. b Binding of AREG fragments to ECM proteins. ELISA plates were coated with ECM proteins and incubated with AREG fragments or GST only. Graphs show signals given when detecting GST. AREG 26-38 is shown in red. n = 3. c AREG 26-38 and AREG 26-38 scramble sequences fused to GST. d Binding of AREG 26-38 and AREG 26-38 scramble to ECM proteins. ELISA plates were coated with ECM proteins and incubated with AREG 26-38 , AREG 26-38 scramble, or GST only. Graphs show signals given when detecting GST. n = 3. For ( b , d ) data are means ± SEM. One-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. *** P < 0.001, otherwise indicated; n.s., non-significant.

Article Snippet: ELISA plates (96-Well Medium Binding, Greiner bio-one) were coated for 1 h at 37 °C with 100 nM solutions of PlGF-2, AREG, NRTN, CXCL-9, CXCL-12 (SDF-1β), CXCL-17, VEGF-A, or BSA (Sigma, A8806).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: NPJ Regenerative Medicine

Article Title: A superior extracellular matrix binding motif to enhance the regenerative activity and safety of therapeutic proteins

doi: 10.1038/s41536-023-00297-0

Figure Lengend Snippet: a Fusion strategy for PDGF-BB (in blue) and IL-1Ra (in purple). AREG 26-38 is represented in red, and N and C indicate the N- and C-terminus, respectively. b Binding affinity of PDGF-BB and IL-1Ra to ECM proteins. ELISA plates were coated with ECM proteins and incubated with PlGF, AREG, or NRTN at increasing concentrations. Bound PlGF, AREG, and NRTN were detected with antibodies. Graphs show the binding affinity ( K D ) obtained from the binding curves. n = 3. c Release kinetics of PDGF-BB and IL-1Ra variants from an ECM-mimetic hydrogel. PDGF-BB or IL-1Ra variants were incorporated in ECM-mimetic hydrogels constituted of fibronectin, vitronectin, tenascin C, fibrinogen and heparan sulfate. Hydrogels were incubated in 10 times volume of buffer, with or without plasmin, that was harvested and replaced every 24 h. Graphs show the cumulative release of PDGF-BB and IL-1Ra variants in buffer. n = 4 per time point. d Retention of PDGF-BB and IL-1Ra following delivery in tissues. IL-1Ra and PDGF-BB variants were injected in mouse dorsal skin or the tibialis anterior muscle. Graphs show the percentage of PDGF-BB and IL-1Ra variants remaining at the site of injection at various time points. n = 4 per time point. e Tumour growth following delivery of PDGF-BB variants at a distal site. Tumour cells were injected subcutaneously. PDGF-BB variants or saline control were injected intradermally 2 cm further at 4 and 6 days after tumour implantation. The graph shows the growth of the tumour over 14 days. n = 6. f Serum concentration of PDGF-BB variants following intradermal delivery. PDGF-BB variant concentrations were measured at different time points after injection. n = 4 per time point. In ( b – f ) data are means ± SEM. For ( b ) two-tailed Student’s t test. For ( c – f ) two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. In ( e ) comparisons are between AREG 26-38 and the other groups. *** P < 0.001, otherwise indicated. n.s., non-significant.

Article Snippet: ELISA plates (96-Well Medium Binding, Greiner bio-one) were coated for 1 h at 37 °C with 100 nM solutions of PlGF-2, AREG, NRTN, CXCL-9, CXCL-12 (SDF-1β), CXCL-17, VEGF-A, or BSA (Sigma, A8806).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Injection, Saline, Control, Concentration Assay, Variant Assay, Two Tailed Test