TNF alpha ELISA Kit

Produktnummer ABIN411361

Produktdetails TNF alpha ELISA Kit

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Bindungsspezifität: AA 77-233

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 15.6 pg/mL - 1000 pg/mL

Untere Nachweisgrenze: 15.6 pg/mL

Applikation: ELISA

Verwendungszweck: Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human TNF alpha

Marke: PicoKine™

Proben: Cell Culture Supernatant, Serum, Plasma (heparin), Plasma (EDTA), Plasma (citrate)

Analytische Methode: Quantitative

Spezifität: Expression system for standard: E.coli,V77-L233

Kreuzreaktivität (Details): There is no detectable cross-reactivity with other relevant proteins.

Sensitivität: <1pg/mL

Benötigtes Material: Microplate reader in standard size. Automated plate washer. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection. Clean tubes and Eppendorf tubes. Washing buffer (neutral PBS or TBS). Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g Nacl

Immunogen: Expression system for standard: E.coli
Immunogen sequence: V77-L233

Anwendungsinformationen

Applikationshinweise: Before using Kit, spin tubes and bring down all components to bottom of tube. Duplicate well assay was recommended for both standard and sample testing.

Kommentare:

Sequence similarities: Belongs to the tumor necrosis factor family.

Plattentyp: Pre-coated

Protokoll: human TNF alpha ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for TNF alpha has been precoated onto 96-well plates. Standards (E.coli,V77-L233) and test samples are added to the wells, a biotinylated detection polyclonal antibody from goat specific for TNF alpha is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the human TNF alpha amount of sample captured in plate.

Testdurchführung:

Aliquot 0.1 mL per well of the 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 15.6pg/mL human TNF alpha standard solutions into the precoated 96-well plate. Add 0.1 mL of the sample diluent buffer into the control well (Zero well). Add 0.1 mL of each properly diluted sample of human cell culture supernates, serum or plasma(heparin, EDTA, citrate) to each empty well. See "Sample Dilution Guideline" above for details. It is recommended that each human TNF alpha standard solution and each sample be measured in duplicate.

Testpräzision:

• Sample 1: n=16, Mean(pg/ml): 93, Standard deviation: 5.1, CV(%): 5.5
• Sample 2: n=16, Mean(pg/ml): 327, Standard deviation: 15.4, CV(%): 4.7
• Sample 3: n=16, Mean(pg/ml): 608, Standard deviation: 31, CV(%): 5.1,
• Sample 1: n=24, Mean(pg/ml): 102, Standard deviation: 7.65, CV(%): 7.5
• Sample 2: n=24, Mean(pg/ml): 319, Standard deviation: 15.3, CV(%): 4.8
• Sample 3: n=24, Mean(pg/ml): 613, Standard deviation: 35, CV(%): 5.7

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (ELISA)

Validierung #102066 (ELISA)

by: Dept. of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School

No.: #102066

Datum: 16.11.2017

Antigen: TNF

Chargennummer: 23913851011

Validierte Anwendung: ELISA

Positivkontrolle: Four cell culture supernates of healthy human Peripheral Blood Mononuclear Cells (hPBMCs) stimulated by T-cell activator (anti CD3/CD28 Antibody). Healthy hPBMCs stimulated by T-cell activator are well known to release inflammatory cytokine such as TNFα and TNFγ. TNFα had been verified using a different ELISA before.

Negativkontrolle: Complete medium using RPMI and human serum (10%)

Bewertung:

Passed. The human TNF ELISA kit ABIN411361 specifically recognizes TNFα in human PBMCs upon stimulation.

Validierungsbilder

TNFα measurements in human PBMCs using ABIN411361. Plate setup and nature of the samples (A). OD450 measurements and calculated concentrations of the utilized standards and controls (B). Standard curve generated using kit standards (C). OD450 measurements (D), calculated concentrations (E), and summary of sample measurements (F).

Protokoll

Full Protocol:

• Harvest hPBMCs at different time points (before irradiation, 4h and 24h after irradiation) by centrifugation. Collect culture media and store them immediately at -20°C.
• Thaw samples on ice followed by centrifugation to remove any residual particulates.
• Reconstitute the human TNFα standard. Shake gently to avoid foaming.
• Prepare the biotinylated anti-Human TNFα antibody working solution 30min prior to the experiment according to the kit manual.
• Prepare the Avidin-Biotin-Peroxidase Complex (ABC) working solution 30min prior to the experiment according to the kit manual and keep it at 37°C until use.
• Keep the TMB color developing agent and TMB stop solution at 37°C for 30min until use.
• Prepare the Washing Buffer (0.01M PBS) according to the kit manual.
• Prepare the standards to make 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml Human TNFα standard solutions: pipette 100µl of each of the standards, controls, and unknown samples into the assigned wells into the precoated 96-well plate. Add 100µl of the sample diluent buffer into the control well (Zero background well). Add 100µl of each sample to each empty well as indicated in figure panel A.
• Seal the plate with a new adhesive cover provided and incubate at 37°C for 90min.
• Remove the cover, discard the plate content, and blot the plate onto paper towels. The wells must not dry at any time.
• Add 100µl of biotinylated anti-Human TNFα antibody working solution into each well.
• Seal the plate with a new adhesive cover provided and incubate at 37°C for 60min.
• Remove the cover, discard the plate content, and wash the plate 3x for 1min with 0.01M PBS. Discard the washing buffer and blot the plate onto paper towels.
• Add 100µl of prepared ABC working solution into each well.
• Seal the plate with a new adhesive cover provided and incubate at 37°C for 30min.
• Wash the plate 5x for 1-2min with 0.01M TBS or 0.01M PBS. Discard the washing buffer and blot the plate onto paper towels.
• Add 90μl of prepared TMB color developing agent into each well.
• Seal the plate with a new adhesive cover and incubate at 37°C in dark for 20min.
• Add 100µl of prepared TMB stop solution into each well. The color changes into yellow immediately.
• Incubate for 10min after adding the stop solution.
• Read the OD absorbance at 450nm in a microplate reader within 30min after adding the stop solution.

Anmerkungen:

• Samples were not diluted since a low target protein concentration was expected.

• The standards and some samples were prepared in duplicates. The negative (n=1) and positive controls (n=4) were prepared in triplicates. The six different spike controls were measured without replicates (Figure panel A for details).

• Increased TNFα release was detected in cell culture supernates stimulated by T cell activator without irradiation except for Sample #3. Low concentration of TNFα in all non-stimulated samples with or without irradiation were observed. We think that sample #3 was possibly a low responder for T cell stimulation (figure panel F).

• Furthermore, in sample #1, TNFα level in cell culture supernate of irradiated cells (C-1) was slightly upregulated at 24 hours after irradiation compared with non-irradiated (C-3) (graph in figure panel F). Given this, although high-dose irradiation was probably enough to suppress TNFα release from the cells stimulated with T cell activator in some cells (samples #2 and #3), TNFα could be released by irradiation in some cells (sample #1).

Handhabung

Handhabung: Avoid multiple freeze-thaw cycles.

Lagerung: -20 °C,4 °C

Informationen zur Lagerung: Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles

Haltbarkeit: 12 months

Referenzen für TNF alpha ELISA Kit (ABIN411361)

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Details zu TNF alpha

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Andere Bezeichnung: TNF (TNF alpha Produkte)

Hintergrund:

Protein Function: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Impairs regulatory T-cells (Treg) function in individuals with rheumatoid arthritis via FOXP3 dephosphorylation. Upregulates the expression of protein phosphatase 1 (PP1), which dephosphorylates the key 'Ser-418' residue of FOXP3, thereby inactivating FOXP3 and rendering Treg cells functionally defective (PubMed:23396208). Key mediator of cell death in the anticancer action of BCG-stimulated neutrophils in combination with DIABLO/SMAC mimetic in the RT4v6 bladder cancer cell line (PubMed:22517918). .

Background: Tumor necrosis factor-alpha(TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer. Human Tumor Necrosis Factor(TNF) and Lymphotoxin(TNF-beta) are cytotoxic proteins which have similar biological activities and share 30 % amino acid homology. TNF-alpha is produced by monocytes, which can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro. TNF is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF has been purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. TNF-alpha has been mapped to human chromosome 6.

Synonyms: Tumor necrosis factor,Cachectin,TNF-alpha,Tumor necrosis factor ligand superfamily member 2,TNF-a,Tumor necrosis factor, membrane form,N-terminal fragment,NTF,Intracellular domain 1,ICD1,Intracellular domain 2,ICD2,C-domain 1,C-domain 2,Tumor necrosis factor, soluble form,TNF,TNFA, TNFSF2,

Full Gene Name: Tumor necrosis factor

Cellular Localisation: Cell membrane, Single-pass type II membrane protein.

Gen-ID: 7124

UniProt: P01375

Pathways: NF-kappaB Signalweg, Apoptose, Caspase Kaskade in der Apoptose, TLR Signalweg, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome