tnf α production by elisa  (R&D Systems)

Journal: mAbs

Article Title: Higher order receptor clustering due to the IgG3 subclass is necessary for TLR4 signaling and tolerance induction by novel human anti-TLR4 antibodies

doi: 10.1080/19420862.2025.2515415

Figure Lengend Snippet: Novel human IgG4 antibodies 3D7, 8G10, and 8H1 recognize TLR4 and MD2 while 7D11 recognizes only MD2. Detection of recombinant TLR4/MD2 heterodimer (a) TLR4 alone (b) or MD2 alone (c) by ELISA using a 3-fold dilution series of human IgG4 antibodies 3D7, 8G10, 8H1, and 7D11. With the concentration of TLR4Ab held constant, an increasing concentration of soluble TLR4 was added in the ELISA assay to compete with the plate bound TLR4/MD2 (d). Data represents a single experiment in duplicate.

Article Snippet: Supernatants were again collected at 24 h and assayed for TNF-α production by ELISA (R&D Systems, #DY210–05).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: mAbs

Article Title: Higher order receptor clustering due to the IgG3 subclass is necessary for TLR4 signaling and tolerance induction by novel human anti-TLR4 antibodies

doi: 10.1080/19420862.2025.2515415

Figure Lengend Snippet: IgG3 TV-7D11 induces tolerance in primary human myeloid cells. Human CD14+ monocytes were purified by magnetic bead negative selection. 1 × 10^6 cells/well were seeded in 24-well plates and stimulated with IgG3 8H1 (10 μg/ml), IgG3 TV-7D11 (10 μg/ml), or LPS (.01 μg/ml). After 24 h supernatants were removed and tested for TNF-α by ELISA (a), followed by addition of fresh media and culture for 5 d to allow cells to return to a resting state (b). On day 6, cells were split into 96-well plates at 50,000 cells/well and allowed to rest for 24 h prior to re-challenge with LPS at .01 μg/ml. Supernatants were again collected at 24 h and assayed for TNF-α production by ELISA (c). Data points represent three individuals from two independent experiments. Statistical analysis by ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Supernatants were again collected at 24 h and assayed for TNF-α production by ELISA (R&D Systems, #DY210–05).

Techniques: Purification, Selection, Enzyme-linked Immunosorbent Assay

Journal: Journal of medical virology

Article Title: Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

doi: 10.1002/jmv.28768

Figure Lengend Snippet: FIGURE 2 VG161 enhances the immune response in breast cancer. (A–C) MCF‐7 cells or MDA‐MB‐231 and SK‐BR‐3 cells were infected with VG161 at a multiplicity of infection of 0.5 or 1 for the indicated times. Human IL‐12p70, IL‐15, and IgG4 secretion levels were measured by enzyme‐linked immunosorbent assays (ELISAs). Then, 50 × 104 EMT‐6 cells were subcutaneously implanted into the second mammary fat pad of both sides of BALB/c mice to establish an orthotopic transplantation model. Tumor‐bearing mice were infected intratumorally with either vehicle (PBS) or 1 × 107 PFU of mVG161 virus on the right side (n = 8 mice per group). Both right and left side tumors were harvested 120 h after the final injection. (D) RNA was isolated and purified followed by library construction and sequencing performed on the BGISEQ‐500 platform by Wuhan Genomic Institution. KEGG analysis of immunostimulatory pathway activation was performed in each group. (E) The mRNA levels of relevant genes in TNF and IL‐17 pathway were measured by RT‐qPCR. (F−G) Mouse serum was collected 120 h after the final injection, then mouse IFN‐γ and TNF‐α levels were assessed by ELISAs. The levels of serum BUN, creatinine, UA, AST, GGT, and ALT were analyzed using the AU680 Chemistry System. (H) Immunohistochemical analysis of CD3+, CD4+, and CD8+ cells was performed on tumor sections. Scale bar = 100 μm. Data are represented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. SD, standard deviation.

Article Snippet: Mice serum was analyzed for Interferon gamma (IFN‐γ) and Tumor Necrosis Factor alpha (TNF‐α) production using the IFN‐γ and TNF‐α Mouse DuoSet ELISA Kit (R&D Systems) according to the manufacturer's recommended procedures.

Techniques: Infection, Transplantation Assay, Virus, Injection, Isolation, Purification, Sequencing, Activation Assay, Quantitative RT-PCR, Immunohistochemical staining, Standard Deviation

Journal: Journal of medical virology

Article Title: Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

doi: 10.1002/jmv.28768

Figure Lengend Snippet: FIGURE 5 VG161 treatment followed by cotreatment with Paclitaxel (PTX) greatly alters the tumor microenvironment (TME). To establish an orthotopic transplantation model, 50 × 104 EMT‐6 cells were subcutaneously implanted into the second mammary fat pad of right side of BALB/c mice. Tumor‐bearing mice were treated with PBS, 1 × 107 PFU of mVG161, PTX (25 mg/kg), or mVG161 + PTX. Tumors were treated with mVG161 for three consecutive days. PTX treatments were every other day for two injections. Tumors were harvested 120 h after the final injection. (A–B) The percentages of different subsets of lymphoid cells, including CD8+ T cells and natural killer cells (NKs) (which expressed IFN‐γ, TNF‐α, and CD107a), and myeloid cells, including m‐MDSCs (which expressed CD11b+/Ly6C+) and DC cells (which expressed MHCII+

Article Snippet: Mice serum was analyzed for Interferon gamma (IFN‐γ) and Tumor Necrosis Factor alpha (TNF‐α) production using the IFN‐γ and TNF‐α Mouse DuoSet ELISA Kit (R&D Systems) according to the manufacturer's recommended procedures.

Techniques: Transplantation Assay, Injection

Journal: Journal of medical virology

Article Title: Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

doi: 10.1002/jmv.28768

Figure Lengend Snippet: FIGURE 6 Schematic depicting the effect of VG161 and Paclitaxel (PTX) in breast cancer (BC). Combining the VG161 oncolytic herpesvirus with PTX can significantly repress BC tumor growth and reduce metastatic pulmonary lesions. Additionally, this combination alters the tumor microenvironment, which is associated with the infiltration of lymphoid cells, including CD4+ T cells, CD8+ T cells, and natural killer cells (expressing TNF‐α and IFN‐γ) and myeloid cells including macrophages, m‐MDSCs, and DC cells. DC, dendritic cell; m‐MDSCs, myeloid‐derived suppressor cells; SD, standard deviation.

Article Snippet: Mice serum was analyzed for Interferon gamma (IFN‐γ) and Tumor Necrosis Factor alpha (TNF‐α) production using the IFN‐γ and TNF‐α Mouse DuoSet ELISA Kit (R&D Systems) according to the manufacturer's recommended procedures.

Techniques: Expressing, Derivative Assay, Standard Deviation