TNF alpha Kit ELISA

N° du produit ABIN1980113

Détail du produit TNF alpha Kit ELISA

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

Reactivité: Porc

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 20 pg/mL-10 ng/mL

Seuil minimal de détection: 20 pg/mL

Application: ELISA

Fonction: Porcine TNF alpha ELISA Kit for cell culture supernatants, plasma, and serum samples.

Type d'échantillon: Plasma, Cell Culture Supernatant, Serum

Analytical Method: Quantitative

Specificité: The sandwich ELISA antibody pair detects Porcine TNF-alpha.

Sensibilité: 20 pg/mL

Attributs du produit:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Ingrédients:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Matériel non inclus:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Information d'application

Indications d'application: Recommended Dilution for serum and plasma samples2 fold

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Préparation des réactifs:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: If your samples need to be diluted, Assay Diluent C (Item L) should be used for dilution of serum/plasma/culture supernatants. Suggested dilution for normal serum/plasma: 2 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
   3. Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 400 µL Assay Diluent C (Item L) into Item C vial to prepare a 40 ng/mL standard solution. Dissolve the powder thoroughly by a gentle mix. Add 200 µL TNF-alpha standard from the vial of Item C, into a tube with 600 µL Assay Diluent C to prepare a 10 ng/mL standard solution. Pipette 400myl Assay Diluent C into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent C serves as the zero standard (0 ng/mL). 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 200 µL standard + 600 µL 10 3.333 1.111 0.370 0.123 0.041 0.014 0 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B (Item E) into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 600-fold with 1x Assay Diluent B (Item E). For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare a 600-fold diluted HRP-Streptavidin solution (don't store the diluted solution for next day use). Mix well.

Procédure de l'essai:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Calcul des résultats:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent C Porcine TNF-alpha concentration (ng/mL) 0.01 0.1 1 10 O D =4 50 n m 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of TNF-alpha is typically less than 20 pg/mL.
Recovery: Recovery was determined by spiking TNF-alpha into porcine serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 111.7 102-121 Plasma 97.02 70-126 Cell culture media 132.1 123-142
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 89.04 94.57 103.3 Range ( %) 78-97 85-102 92-118 1:4 Average % of Expected 75.78 74.79 76.55 Range ( %) 67-85 67-86 68-88
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Précision du teste: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Avoid repeated freeze-thaw cycles.

Stock: -20 °C

Stockage commentaire: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Date de péremption: 6 months

Références pour TNF alpha Kit ELISA (ABIN1980113)

Zong, Song, Wang, Xia, Hu, Han, Wang: "LFP-20, a porcine lactoferrin peptide, ameliorates LPS-induced inflammation via the MyD88/NF-κB and MyD88/MAPK signaling pathways." dans: Developmental and comparative immunology, Vol. 52, Issue 2, pp. 123-31, (2015) (PubMed).

Zhang, Hou, Zhong, Li, Chen, Li, Wen, Wang, Liu, Zhong: "Porcine reproductive and respiratory syndrome virus activates inflammasomes of porcine alveolar macrophages via its small envelope protein E." dans: Virology, Vol. 442, Issue 2, pp. 156-62, (2013) (PubMed).

Galindo, Ayllón, Smrdel, Boadella, Beltrán-Beck, Mazariegos, García, de la Lastra, Avsic-Zupanc, Kocan, Gortazar, de la Fuente: "Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection." dans: Parasites & vectors, Vol. 5, pp. 181, (2012) (PubMed).

Détail du antigène

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

Autre désignation: TNF-alpha (TNF alpha Produits)

Synonymes: DIF Kit ELISA, TNF-alpha Kit ELISA, TNFA Kit ELISA, TNFSF2 Kit ELISA, RATTNF Kit ELISA, Tnfa Kit ELISA, tnf Kit ELISA, TNF-a Kit ELISA, TNFalpha Kit ELISA, Tnfsf1a Kit ELISA, TNFa Kit ELISA, cTNF Kit ELISA, Tnf-alpha Kit ELISA, tnfa-like Kit ELISA, TNF-ALPHA Kit ELISA, dif Kit ELISA, tnfa Kit ELISA, xtnf Kit ELISA, tnfsf2 Kit ELISA, tnf-alpha Kit ELISA, Cachectin Kit ELISA, tumor necrosis factor Kit ELISA, tumor necrosis factor b (TNF superfamily, member 2) Kit ELISA, tumor necrosis factor alpha Kit ELISA, tumor necrosis factor a (TNF superfamily, member 2) Kit ELISA, TNF Kit ELISA, Tnf Kit ELISA, tnf Kit ELISA, tnfb Kit ELISA, tnf-alpha Kit ELISA, LOC103694380 Kit ELISA, tnfa Kit ELISA

Sujet: Tumor necrosis factor (Cachectin) (TNF-alpha) (Tumor necrosis factor ligand superfamily member 2) (TNF-a)

ID gène: 397086

UniProt: P23563

Pathways: Signalisation NF-kappaB, Apoptose, Caspase Cascade in Apoptosis, Signalisation TLR, 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