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Human TNF ELISA Set

Human TNF ELISA Set

(RUO)
Human TNF ELISA Set

This standard curve is fordemonstration only. A standard curve must be run with each assay. \"Typical Standard Curve\" and 20-plate yield were obtained in the BD Biosciences Pharmingen laboratory, using the recommended procedure and manual plate washing.

This standard curve is fordemonstration only. A standard curve must be run with each assay. \"Typical Standard Curve\" and 20-plate yield were obtained in the BD Biosciences Pharmingen laboratory, using the recommended procedure and manual plate washing.

Product Details
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BD OptEIA™
Human (QC Testing)
ELISA (Routinely Tested)
RUO
AB_2869042


Description

    The OptEIA™ Set for human tumor necrosis factor (TNF, formerly knowns as TNF-α) contains the components necessary to develop enzyme-linked immunosorbent assays (ELISA) for natural or recombinant human TNF in serum, plasma, and cell culture supernatants. Sufficient materials are provided to yield approximately 20 plates of 96-wells if the recommended storage, materials, buffer preparation, and assay procedure are followed as specified in this package.  

  

  

  

    Assay Optimization  

  

    BD OptEIA™ Sets allow flexible assay design to fit individual laboratory needs. To design an immunoassay with different sensitivity and dynamic range, the following parameters can be varied: Capture, Detection Antibody titers, Incubation time, Incubation temperature, Assay Diluent formulation, Buffer pH, ionic strength, protein concentration, Type of substrate, Washing technique (i.e., number of wash repetitions and soak times).  

  

  

  

    Standardization: The standard in this Set is calibrated against purified baculovirus expressed recombinant human TNF produced at BD Biosciences Pharmingen. The NIBSC/WHO International Standard 87/650 (recombinant human TNF expressed in E. Coli ) was evaluated in this Set. The conversion factor for NIBSC material is as follows: 1 μg NIBSC 87/650 TNF-α = 1.14 μg BD OptEIA™ TNF.  

Preparation And Storage

Store unopened reagents at 2-8°C. do not use reagents after expiration date, or if turbidity is evident. Before use, bring all reagents to room temperature (18-25°C). Immediately after use, return to proper storage conditions. Lyophilized standards are stable until expiration date. After reconstitution, immediately aliquot standard stock in polypropylene vials at 50 µl per vial anf freeze at -80°C for up to 6 months. If necessary, store at 2-8°C for up to 8 hours prior to aliquotting/freezing. Do not leave reconstituted standard at room temperature.

Recommended Assay Procedures

    Recommended buffers, solutions   

  

    Note: Do not use sodium azide in these preparations. Sodium azide inactivates the horseradish peroxidase enzyme.  

  

    The BD OptEIA™ Reagent Set B (Cat. No 550534) containing Coating Buffer, Assay Diluent, Substrate Reagents A and B, Stop Solution and 20X Wash Buffer Concentrate is recommended.  

  

    1. Coating Buffer - 0.1 M Sodium Carbonate, pH 9.5; 7.13 g NaHCO3, 1.59 g Na2CO3; q.s. to 1.0 L; pH to 9.5 with 10 N NaOH.  

  

    Freshly prepare or use within 7 days of preparation, stored at 2-8°C.   

  

    2. Assay Diluent- PBS* with 10% FBS#, pH 7.0. The BD Pharmingen™ Assay Diluent (Cat. No. 555213) is recommended.   

  

    *Phosphate-Buffered Saline: 80.0 g NaCl.11.6 g Na2HPO4, 2.0 g KH2PO4, 2.0 g KCL, q.s. to 10 L; pH to 7.0.  

  

    #Fetal Bovine Serum: Hyclone Cat. No. SH30088 (heat-inactivated) recommended.  

  

    Freshly prepare or use within 3 days of preparation, with 2-8°C  storage.  

  

    3. Wash Buffer - PBS* with 0.05% Tween-20.   

  

    4. Substrate Solution - Tetramethylbenzidine (TMB) and Hydrogen Peroxide. The BD Pharmingen™ TMB Substrate Reagent Set (Cat. No. 555214) is recommended.  

  

    5. Stop Solution - 1 M H3PO4 or 2 N H2SO4  

  

  

  

    Additional Materials Required   

  

    1. 96-well BD Falcon™ ELISA plates (Cat. No. 353279) are recommended  

  

    2. Microplate reader capable of measuring absorbance at 450 nm  

  

    3. Precision pipettes   

  

    4. Graduated cylinder, one liter  

  

    5. Deionized or distilled water  

  

    6. Wash bottle or automated washer  

  

    7. Log-log graph paper or automated data reduction  

  

    8. Tubes to prepare standard dilutions  

  

    9. Laboratory timer  

  

    10. Plate sealers or parafilm  

  

  

  

    Specimen Collection and Handling: Specimens should be clear, non-hemolyzed and non-lipemic.  

  

    Cell culture supernatants: Remove any particulate material by centrifugation and assay immediately or store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles.  

  

    Serum: Use a serum separator tube and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles.  

  

    Plasma: Collect plasma using citrate, EDTA, or heparin as anticoagulant. Centrifuge for 10 minutes at 1000 x g within 30 minutes of collection. Assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles.  

  

  

  

    Standards Preparation and Handling   

  

    1. Reconstitution: After warming lyophilized standard to room temperature, carefully open vial to avoid loss of material. Reconstitute lyophilized standard with 1.0 mL of deionized water to yield a stock standard. Allow the standard to equilibrate for at least 15 minutes before making dilutions. Vortex gently to mix.  

  

    2. Storage/ handling of reconstituted standard: After reconstitution, immediately** aliquot standard stock in polypropylene vials at 50 μl per vial and freeze at -80°C  for up to 6 months. **If necessary, store at 2-8° C for up to 8 hours prior to aliquotting/freezing. (Do not leave reconstituted standard at room temperature.)   

  

    3. Standards Preparation for Assay:   

  

    a. Prepare a 500 pg/mL standard from the stock standard. Vortex to mix. (See dilution instructions on Instruction/Analysis Certificate.)   

  

    b. Add 300 μL Assay Diluent to 6 tubes. Label as 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, 15.6 pg/mL, and 7.8 pg/mL.   

  

    c. Perform serial dilutions by adding 300 μL of each standard to the next tube and vortexing between each transfer. Assay Diluent serves as the zero standard (0 pg/mL).  

  

  

  

    Working Detector Preparation  

  

    (Note: One-step incubation of Biotin/Streptavidin reagents.) Add required volume of Detection Antibody to Assay Diluent. Within 15 minutes prior to use, add required quantity of Enzyme Reagent, vortex or mix well. For recommended dilutions, see lot-specific Instruction/Analysis Certificate. For a full 96-well plate, prepare 12 mL of Working Detector. Discard any remaining Working Detector after use.  

  

  

  

    Detailed Assay Procedure  

  

    1. Coat microwells with 100 μL per well of Capture Antibody diluted in Coating Buffer. For recommended antibody coating dilution, see lot-specific Instruction/Analysis Certificate. Seal plate and incubate overnight at 4° C.   

  

    2. Aspirate wells and wash 3 times with ≥ 300 μL/well Wash Buffer. After last wash, invert plate and blot on absorbent paper to remove any residual buffer.  

  

    3. Block plates with ≥ 200 μL/well Assay Diluent. Incubate at RTfor 1 hour.  

  

    4. Aspirate/wash as in step 2.  

  

    5. Prepare standard and sample dilutions in Assay Diluent. See "Standards Preparation and Handling."  

  

    6. Pipette 100 μL of each standard, sample, and control into appropriate wells. Seal plate and incubate for 2 hours at RT.  

  

    7. Aspirate/ wash as in step 2, but with 5 total washes.  

  

    8. Add 100 μL of Working Detector (Detection Antibody + SAv-HRP reagent) to each well. Seal plate and incubate for 1 hour at RT.  

  

    9. Aspirate/ wash as in step 2, but with 7 total washes. NOTE: In this final wash step, soak wells in wash buffer for 30 seconds to 1 minute for each wash.  

  

    10. Add 100 μL of Substrate Solution to each well. Incubate plate (without plate sealer) for 30 minutes at room temperature in the dark.  

  

    11. Add 50 μL of Stop Solution to each well.  

  

    12. Read absorbance at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, subtract absorbance at 570 nm from absorbance 450 nm.  

  

  

  

    Assay Procedure Summary  

  

    1. Add 100 μL diluted Capture Ab to each well. Incubate overnight at 4° C.  

  

    2. Aspirate and wash 3 times.  

  

    3. Block plates: 200 μL Assay Diluent to each well. Incubate 1 hr RT.  

  

    4. Aspirate and wash 3 times.  

  

    5. Add 100 μL standard or sample to each well. Incubate 2 hr RT.  

  

    6. Aspirate and wash 5 times.  

  

    7. Add 100 μL Working Detector (Detection Ab + SAv-HRP) to each well. Incubate 1 hr RT  

  

    8. Aspirate and wash 7 times (with 30 sec to 1 min soaks).  

  

    9. Add 100 μL Substrate Solution to each well. Incubate 30 min RT in dark.  

  

    10. Add 50 μL Stop Solution to each well. Read at 450 nm within 30 min with λ correction 570 nm.  

  

  

  

    Calculation of Results  

  

    Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.  

  

    Plot the standard curve on log-log graph paper, with TNF concentration on the x-axis and absorbance on the y-axis. Draw the best fit curve through the standard points.  

  

    To determine the TNF concentration of the unknowns, find the unknown's mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the TNF concentration. If samples were diluted, multiply the TNF concentration by the dilution factor.   

  

    Computer data reduction may also be employed, utilizing log-log regression analysis.  

  

  

  

    Specificity  

  

    Cross Reactivity: The following factors were tested in the BD OptEIA™ assay at ≥ 100 ng/mL (except where noted) and no cross-reactivity (value ≥ 4 pg/mL) was identified.  

  

    Recombinant Human: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 (p40), IL-12 (p70), IL-13, IL-15, eotaxin, G-CSF (10ng/mL), GM-CSF, GKO, IFN-γ, CD23, Lymphotactin (10ng/mL), MIP-1β, MCP-1, MCP-2, MCP-3, 4, NAP2, IP-10, NT-3, PDGF-AA, SCF (10ng/mL), LT-α (TNF-β), VEGF  

  

    Recombinant Mouse: IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p70), IL-15, IFN-γ, GM-CSF, MCP-1, TCA3, TNF   

  

    Recombinant Rat: IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF, RANTES (10ng/mL)   

  

    Other: Viral IL-10 (10 ng/mL), Rabbit TNF (10ng/mL)  

  

  

  

    Limitations of the Procedure  

  

    · Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-assayed.  

  

    · Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.  

  

    · BD OptEIA™ Sets are intended for use as an integral unit. Do not mix reagents from different Set batches. Reagents from other manufacturers are not recommended for use in this Set.  

Product Notices

  1. For online training for BD OptEIA™ Set ELISA Techniques, please refer to http://www.bdbiosciences.com/OptEIA/downloads.shtml
  2. Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-assayed.
  3. Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.
  4. BD OptEIA™ Sets are intended for use as an integral unit. Do not mix reagents from different Set batches. Reagents from other manufacturers are not recommended for use in this Set.
  5. Reagents which contain preservatives may be toxic if ingested, inhaled, or in contact with skin.
  6. Handle all serum and plasma specimens in accordance with NCCLS guidelines for preventing transmission of blood-borne infections.
  7. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. ProClin is a trademark of Rohm and Haas Company.
555212 Rev. 1
Components
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Description Quantity/Size Part Number
Capture Antibody Purified Anti-Human TNF N/A 51-26371E
Detection Antibody Biotin Anti-Human TNF N/A 51-26372E
Recombinant Human TNF Lyophilized Standard N/A 51-26376E
Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) N/A 51-9002813
555212 Rev. 1
Citations & References
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Development References (10)

  1. Black RA, Rauch CT, Kozlosky CJ, et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997; 385(6618):729-733. (Biology). View Reference
  2. Danis VA, Franic GM, Rathjen DA, Brooks PM. Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes. Clin Exp Immunol. 1991; 85(1):143-150. (Biology). View Reference
  3. Hogan MM, Vogel SN. Production of tumor necrosis factor by rIFN-gamma-primed C3H/HeJ (Lpsd) macrophages requires the presence of lipid A-associated proteins. J Immunol. 1988; 141(12):4196-4202. (Biology). View Reference
  4. Jaattela, M. . Biologic activities and mechanisms of action of tumor necrosis factor-α/cachectin. Lab Invest. 1991; 64:724-742. (Biology).
  5. Kriegler M, Perez C, DeFay K, Albert I, Lu SD. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifications for the complex physiology of TNF. Cell. 1988; 53(1):45-53. (Biology). View Reference
  6. Petyovka N, Lyach L, Voitenok NN. Homologous ELISA for detection of oligomeric human TNF: properties of the assay. J Immunol Methods. 1995; 186(2):161-170. (Biology). View Reference
  7. Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Biology). View Reference
  8. Rathjen DA, Cowan K, Furphy LJ, Aston R. Antigenic structure of human tumour necrosis factor: recognition of distinct regions of TNF alpha by different tumour cell receptors. Mol Immunol. 1991; 28(1-2):79-86. (Biology). View Reference
  9. Smith RA, Baglioni C. The active form of tumor necrosis factor is a trimer. J Biol Chem. 1987; 262(15):6951-6954. (Biology). View Reference
  10. Wang AM, Creasey AA, Ladner MB, et al. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science. 1985; 228(4696):149-154. (Biology). View Reference
View All (10) View Less
555212 Rev. 1

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.