Mouse TNF ELISA Set II(RUO)
The OptEIA™ Set for mouse tumor necrosis factor (TNF ) contains the components necessary to develop enzyme-linked immunosorbent assays (ELISA) for natural or recombinant mouse 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.
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: This immunoassay is calibrated against purified Baculovirus-expressed recombinant mouse TNF during product development.
Preparation And Storage
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 , 3.56 g Na 2CO3; q.s. to 1.0 L; pH to 9.5. 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. Freshly prepare or use within 3 days of preparation, stored at 2-8°C.
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. Note the amount of protein indicated on the lyophilized standard vial label and record the reconstituted standard concentration below for future use. 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 labeled with the correct protein concentration 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 1000 pg/mL standard from the stock standard. Vortex to mix.
b. Add 300 µL Assay Diluent to 6 tubes. Label as 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, and 15.6 pg/mL.
Detailed Assay Procedure
1. Dilute the Capture Antibody 1:250 in Coating Buffer and coat microwells with 100 µl of diluted Capture Antibody per well. Seal plate and incubate overnight at 4°C. Do not dilute more Capture Antibody than is needed for your experiment.
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 room temperature (RT) for 1 hour.
4. Aspirate/wash as in step 2.
5. Prepare standard and sample dilutions in Assay Diluent. See Standards Preparation and Handling. Be sure to record the reconstituted standard concentration for future use.
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. Dilute the Detection Antibody 1:250 in Assay Diluent and add 100 µl of diluted Detection Antibody to each well. Seal plate and incubate for 1 hour at RT. Do not dilute more Detection Antibody than is needed for your experiment.
9. Aspirate/ wash as in step 2, but with 5 total washes.
10. Dilute the Enzyme Reagent (SAv-HRP) 1:250 in Assay Diluent and add 100 µl of diluted Enzyme Reagent to each well. Seal plate and incubate for 30 minutes at RT. Do not dilute more Enzyme Reagent than is needed for your experiment.
11. 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.
12. Add 100 µL of TMB Substrate Solution to each well. Incubate plate (without plate sealer) for 30 minutes at room temperature in the dark.
13. Add 50 µL of Stop Solution to each well.
14. 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 diluted Detection Ab to each well. Incubate 1 hr RT.
8. Aspirate and wash 5 times.
9. Add 100 µL diluted SAv-HRP to each well. Incubate 30 min RT.
10. Aspirate and wash 7 times (with 30 sec. to 1 min soaks).
11. Add 100 µL TMB Substrate Solution to each well. Incubate 30 min RT in dark.
12. 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.
Cross Reactivity: The following factors were tested in the BD OptEIA™ assay at ≥ 10 ng/mL and no cross-reactivity (value ≥ 15 pg/mL) was identified.
Recombinant Human: TNF
Recombinant Mouse: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, GM-CSF, IFN-γ, LT-α, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, sTNFRI, sTNFRII, RANTES
Recombinant Rat: IL-1α, IL-2, IL-4, IL-6, IL-10, IL-18, GM-CSF, IFN-γ
Limitations of the Procedure
-Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-tested.
-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.
- For online training for BD OptEIA™ Set ELISA Techniques, please refer to http://www.bdbiosciences.com/OptEIA/downloads.shtml
- 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.
- Reagents which contain preservatives may be toxic if ingested, inhaled, or in contact with skin.
- ProClin is a trademark of Rohm and Haas Company.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Handle all serum and plasma specimens in accordance with NCCLS guidelines for preventing transmission of blood-borne infections.
|Description||Quantity/Size||Part Number||EntrezGene ID|
|Capture Antibody Purified Anti-Mouse TNF||N/A||51-9004717||N/A|
|Detection Antibody Biotin Anti-Mouse TNF||N/A||51-9004718||N/A|
|Recombinant Mouse TNF Lyophilized Standard||N/A||51-26986E||N/A|
|Enzyme Reagent Streptavidin-HRP (SAv-HRP) 250X||N/A||51-9004787||N/A|
Development References (13)
Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Biology). View Reference
Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Biology). View Reference
Drew PD, Chavis JA. Female sex steroids: effects upon microglial cell activation. J Neuroimmunol. 2000; 111(1-2):77-85. (Biology). View Reference
Drew PD, Chavis JA. Inhibition of microglial cell activation by cortisol. Brain Res Bull. 2000; 52(5):391-396. (Biology). View Reference
Drew PD, Chavis JA.. The cyclopentone prostaglandin 15-deoxy-Delta(12,14) prostaglandin J2 represses nitric oxide, TNF-alpha, and IL-12 production by microglial cells.. J Neuroimmunol. 2001; 115(1-2):28-35. (Biology). View Reference
Ferran C, Dautry F, Merite S, et al. Anti-tumor necrosis factor modulates anti-CD3-triggered T cell cytokine gene expression in vivo. J Clin Invest. 1994; 93(5):2189-2196. (Biology). View Reference
He J, Gurunathan S, Iwasaki A, Ash-Shaheed B, Kelsall BL. Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses . J Exp Med. 2000; 191(9):1605-1610. (Biology). View Reference
Leiby DA, Fortier AH, Crawford RM, Schreiber RD, Nacy CA. In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies. Infect Immun. 1992; 60(1):84-89. (Biology). View Reference
Merrick BA, He CY, Craig WA, et al. Two dimensional gel electrophoresis of cellular and secreted proteins from rat alveolar macrophages after lipopolysaccharide treatment. Appl Theor Electrophor. 1992; 2(6):177-187. (Biology). View Reference
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
Rabinovici R, Bugelski PJ, Esser KM, et al. Tumor necrosis factor-alpha mediates endotoxin-induced lung injury in platelet activating factor-primed rats. J Pharmacol Exp Ther. 1993; 267(3):1550-1557. (Biology). View Reference
Sheehan KC, Ruddle NH, Schreiber RD. Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors. J Immunol. 1989; 142(11):3884-3893. (Biology). View Reference
Takahashi S, Kapas L, Fang J, Krueger JM. An anti-tumor necrosis factor antibody suppresses sleep in rats and rabbits. Brain Res. 1995; 690(2):241-244. (Biology). View Reference
Please refer to Support Documents for Quality Certificates
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.