14-7178-81

antibody from Invitrogen Antibodies

Targeting: IL17A CTLA8, IL-17, IL-17A, IL17

Provider product page for 14-7178-81

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Antibody Data
Antigen structure
References [40]
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Product number: 14-7178-81 - Provider product page
Provider: Invitrogen Antibodies
Product name: IL-17A Monoclonal Antibody (eBio64CAP17), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The eBio64CAP17 antibody reacts with human IL-17A; the antibody has been reported to cross react with Rhesus monkey IL-17A, as verified by intracellular staining experiments. The eBio64CAP17 antibody is a neutralizing antibody. This antibody has been shown to have no reactivity to human IL-17F. Reactivity to other members of the IL-17 family has not been evaluated. Interleukin-17A (IL-17A) is a CD4+ T cell-derived cytokine that promotes inflammatory responses in cell lines and is elevated in rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, and transplant rejection. The cDNA encoding human IL-17A was isolated from a library of CD4+ T cells; the encoded protein exhibits 72 percent amino acid identity with HVS13 , an open reading frame from a T lymphotropic Herpesvirus saimiri, and 63 percent with mouse CTLA-8 (cytotoxic T-lymphocyte associated antigen-8). Human IL-17A exists as glycosylated 20-30 kD homodimers. High levels of IL-17A homodimer are produced by activated peripheral blood CD4+ T-cells. IL-17A enhances expression of the intracellular adhesion molecule-1 (ICAM-1) in human fibroblasts. Human IL-17A also stimulates epithelial, endothelial, or fibroblastic cells to secrete IL-6, IL-8, G-CSF, and PGE2. In the presence of human IL-17A, fibroblasts can sustain the proliferation of CD34+ hematopoietic progenitors and induce maturation into neutrophils. Mouse, rat, and human IL-17A can induce IL-6 secretion in mouse stromal cells, indicating that all homologs can recognize the mouse IL-17A receptor. IL-23-dependent, IL-17A-producing CD4+ T cells (Th-17 cells) have been identified as a unique subset of Th cells that develops along a pathway that is distinct from the Th1- and Th2- cell differentiation pathways. The hallmark effector molecules of Th1 and Th2 cells, e.g., IFN-g and IL-4, have each been found to negatively regulate the generation of these Th-17 cells. Additionally, activated human CD4+ T cells have been found to produce the IL-17A/F heterodimer, as well as the corresponding homodimers. In comparing the relative potency of IL-17A, IL-17F, and IL-17A/F, all three were found to induce GRO-a secretion; IL-17A was most potent, followed by IL-17A/F heterodimer, then IL-17F (100fold lower than IL-17A). eBio64CAP17 can be used to detect IL-17 heterodimers by immunoprecipitation followed by immunoblot withH17F10A7 anti-IL17F monoclonal antibody. The eBio64CAP17 has been shown to react to rhesus and marmoset primates. Applications Reported: This eBio64CAP17 antibody has been reported for use in intracellular staining followed by flow cytometric analysis, immunoprecipitation, IHC frozen, ELISA, and ELISPOT. (Fluorochrome conjugated eBio64CAP17 is recommended for use in intracellular flow cytometry). Applications Tested: The Affinity Purified eBio64CAP17 antibody has been tested as the capture antibody in a sandwich ELISA for measurement of human IL-17A protein levels, in combination with the biotinylated eBio64DEC17 antibody (13-7179) for detection and recombinant human IL-17A (14-8179) as the standard. A suitable range of concentrations of this antibody for ELISA capture is 0.5 - 2.0 µg/mL. A standard curve consisting of doubling dilutions of the recombinant standard over the range of 1000 pg/mL - 8 pg/mL should be included in each ELISA plate. The Functional Grade Purified eBio64CAP17 antibody has been tested by LAL assay to verify lowest endotoxin levels and has been tested for neutralization of IL-17A bioactivity and for ELISPOT capture. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: eBio64CAP17
Vial size: 50 µg
Concentration: 0.5 mg/mL
Storage: 4° C

IL17A protein structure - 14-7178-81 shown in red.

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Experimental details: Fig 5 Representative flow cytometry plots of cytokine expression by CD4 T cells. CD4 T cells expressing IFN-gamma, IL-2, TNF-alpha and/or IL-17 upon stimulation with BCG or ESAT-6/CFP-10 peptide pools for 12 hours, compared to an unstimulated control sample. The plots represent cytokine-positive cells (red) overlayed onto the background of the entire CD4 T cell parent population (black). Similar assessment was also performed for CD8 and gammadelta T cells (data not shown).

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Experimental details: Figure 3 Presence of IL-17-producing cells in the degenerated IVD tissues. Fifty IVD tissue samples (Group P, n = 20 and Group E, n = 30) from disc degeneration patients and 3 from scoliosis patients were analyzed. In Group P or the control group, there were few or no positive cells (data not shown). The representative results of group E are shown. (a), IL-17-producing cells were detected by a rabbit anti-IL-17 polyclonal antibody and a mouse anti-CD4 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. (b), surface CCR6 expression on the cells was detected with a rabbit anti CD4 monoclonal Ab and a mouse anti-CCR6 monoclonal Ab, followed by secondary staining with an Alexa 488-conjugated donkey anti-rabbit and an Alexa 568-conjugated donkey anti-mouse IgG. DAPI mounting medium was used for nuclear staining. In the top panel, green and red represents the expression of IL-17 and CD4, respectively, and the double-stained cells represent the IL-17-producing cells. In the bottom panel, green and red represents the expression of CD4 and CCR6, respectively, and the double-stained cells demonstrate the surface expression of CCR6 on T lymphocytes.

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Experimental details: Figure 5 Inhibition of RUNX3 regulates the frequencies of Th17 and Th22 in CD4 + T cells from patients with psoriasis. The frequencies of Th17 and Th22 cells in CD4 + T cells from patients with psoriasis transfected with RUNX3 siRNA or an siRNA control was detected using flow cytometry. (A and B) Flow cytometric analysis of Th17 in CD4 + T cells from patients with psoriasis. The percentages of cells in the Q2 region represent the percentage of Th17 cells. (C and D) Flow cytometric analysis of Th22 in CD4 + T cells from patients with psoriasis. The percentages of cells in the Q2 region represent the percentage of Th22 cells. (E) Percentages of Th17 and Th22 cells in CD4 + T cells from patients with psoriasis transfected with RUNX3 siRNA or an siRNA control. Data are presented as the mean +- standard deviation of three experiments. * P

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Experimental details: Figure 7 Circulating percentages of Th17 cells and CCR6-positive cells in peripheral blood are increased in IVD degenerated patients when compared with controls. Heparinized peripheral whole blood cells from 20 patients and 15 healthy controls were stimulated with phorbol myristate acetate (PMA), ionomycin, and monensin for 4 h and subsequently stained with fluorochrome-labeled antibodies as described in Materials and Methods. A(a) Lymphocytes were gated by flow cytometry. A(b) CD3 + T subsets were gated by flow cytometry; the plots in the inset box represent the CD3 + T cells. A(c) Representative IL-17 expression levels in the CD3 + CD8 - T subsets (CD4 + T subsets) from each group are shown. The percentages of positive cells are shown in the upper left panels. A(d) Representative surface CCR6 expression levels on the CD3 + CD8 - IL-17 + subsets from each group are shown. The percentages of positive cells are shown in the right panel. (B) The percentage of circulating Th17 cells was significantly higher in IVD degenerated patients (2.973+-0.689%) than in the control group (1.039+-0.156%; *, p

Supportive validation

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Experimental details: Figure 4 FABP4 promotes Th17 differentiation of naive T cells via increasing IL-17A/IL-23 release (A) The mRNA levels of cytokines were determined by qRT-PCR. (B) The protein level of FABP4 was detected by western blot. (C) The IL-17A and IL-23 levels in cell culture supernatant were assessed by ELISA. (D) The protein levels of NLRP3 inflammasome components were determined by western blot. Naive T cells were cultured under Treg or Th17 cell-polarizing conditions with or without primary macrophages-CM and neutralizing antibody. (E-H) The percentage of Tregs (E and F) or Th17 cells (G and H) was assessed by FACS. GAPDH and beta-actin were used for normalization in qRT-PCR and western blot, respectively. Data are expressed as representative images or the mean +- SD of n = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Supportive validation

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Experimental details: Figure 6 HLA-blocking antibodies inhibit the production of alloreactive memory-CD8 T cells during allogeneic-antigen stimulation. XF-ADSCs were cultured with CD3 T cells and Td-PBMCs via the direct pathway, after 3 weeks whole cells were harvested and memory-CD8 T cells were analyzed by flow cytometry. ( A ) Scheme for the flow cytometry analysis. ( B ) CD3 T cells were cultured for 3 weeks with or without neutralizing or blocking antibodies during allogeneic-antigen stimulation. A combination of anti-human IL-17A and anti-human IFN-gamma antibodies was used to neutralize pro-inflammatory cytokines. A combination of anti-HLA-ABC, anti-HLA-DR and anti-HLA-DQ antibodies was used to block HLAs. The dot plots showed the population of memory-CD8 T cells on the 21st day after allogeneic-antigen stimulation, compared with the control. ( C ) Graphical representation of the number of CFSE-low memory-CD8 T cells corresponding to (B) ( n = 7 (each CD8 TEM), n = 6 (each CD8 TCM)). The control does not contain XF-ADSCs and consist only of CD3 T cells and Td-PBMCs from healthy donor. The data were repeated at least three times; *, p < 0.05; **, p < 0.01; ***, p < 0.001. alpha-IFN+alpha-IL17: combination of anti-human IFN-gamma and anti-human IL-17A antibodies; alpha-HLAs: combination of anti-HLA-ABC, HLA-DR and anti-HLA-DQ antibodies.

Supportive validation

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Experimental details: Fig. 5 Human leukocyte antigens (HLAs) on allogeneic adipose-derived mesenchymal stem cells grown in xeno-free medium (XF-ADSCs) are the major cause of T cell-mediated cytotoxicity. a XF-ADSCs were subjected to allogeneic antigen stimulation for 21-24 days with antibodies against interferon-gamma (IFN-gamma), interleukin (IL)-17A, HLA-ABC, HLA-DR, and HLA-DQ (all at 1 mug/ml) in xeno-free medium containing 5% autologous serum. The effects of the XF-ADSCs with antibodies on T cell-mediated cytotoxicity were evaluated in comparison with those of the XF-ADSCs not containing antibodies. For cell death analysis, the collected XF-ADSCs were stained with anti-CD73 and anti-CD90 antibodies, annexin V, and propidium iodide (PI), and the cell death rate was analyzed by flow cytometry. b Graphical representation of XF-ADSC death corresponding to ( a ) ( n = 21 (untreated), n = 7 (for each sample)); *** p < 0.001. Td-PBMCs T cell-depleted T cell-depleted peripheral blood mononuclear cells