Contents: Human/Mouse TGF-beta1 ELISA Ready-SET-Go!
Catalog Number: 88-7344
Sensitivity: 60 pg/ml
Standard Curve Range: 60 pg/ml - 8000 pg/ml
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Questions? Please consult our answers to frequently asked questions at
http://www.ebioscience.com/faq.
Description
Transforming Growth Factor-beta (TGF-β) is a pleiotropic cytokine which exists in five isoforms, known as TGF-β1-5, with homologies of 70-80% and no homology to TGF-α. TGF-β1 is the most abundant form in lymphoid organs and is found almost ubiquitously while other isoforms are expressed in a more restricted distribution. The biologically active forms of all isoforms are disulfide-linked homodimers. The heat- and acid- stable monomeric subunits have a length of 112 amino acids. The isoforms of TGF-β arise by proteolytic cleavage of longer precursors; the isoforms are derived from the carboxyterminal ends of these precursors. Isoforms isolated from different species are evolutionarily closely conserved and have sequence identities on the order of 98%. Mature human, porcine, simian, chicken and bovine TGF-β1 are identical and differ from mouse TGF-β1 in a single amino acid.
TGF-β1 is produced in very high levels by platelets. Other cellular sources of TGF-β1 include macrophages, lymphocytes, endothelial cells, chondrocytes, and leukemic cells. TGF-β1 secretion can be induced by steroids, retinoids, EGF, NGF, vitamin D3, and IL-1. Activities of TGF-β1 include inhibition of cell growth for inhibitor for normal and transformed epithelial cells, endothelial cells, fibroblasts, neurons, and lymphoid cells and other hematopoietic cell types. TGF-β1 inhibits the proliferation of T cells and NK cells and downregulates the activities of activated macrophages. TGF-β1 blocks the anti-tumor activity of IL-2 – bearing lymphokine-activated killer (LAK) cells.
Recently, TGF-β1 has been found to have a critical role in the development of regulatory T cells. Dendritic cells exposed to tumors have been reported to secrete TGF-β1 and stimulate expansion of naturally-occurring T reg cells. Moreover, TGF-β1 has been shown to act as a costimulatory factor for expression of Foxp3, leading to the differentiation of CD4+CD25+ Treg cells from peripheral CD4+CD25- progeny. TGF-β-induced regulatory T cells have been termed Ti-Treg.
This Mouse/Human TGF-β1 Ready-SET-Go! ELISA Set contains the necessary reagents, standards, buffers and diluents for performing quantitative enzyme-linked immunosorbent assays (ELISA). This ELISA set is specifically engineered for accurate and precise measurement of mouse or human TGF-β1 protein levels from samples including serum, plasma, and supernatants from cell cultures. The calibration standard is highly purified, bioactive recombinant human TGF-β1 (mature form).
Note: This sandwich ELISA recognizes the mature/active form of TGF-β1. Samples (but not standards) should be acid-treated and then neutralized to activate the latent TGF-β1 to the immunoreactive form. Samples should be tested in the ELISA immediately after acid treatment and neutralization. See 'Experimental Procedure'.
Components
- Capture Antibody: Pre-titrated, purified antibody, clone eBioTB2F
- Detection Antibody: Pre-titrated, biotin-conjugated antibody, clone eBio16TFB
- Standard: Recombinant cytokine for generating standard curve and calibrating samples
- ELISA/ELISPOT Coating Buffer Powder: Reconstitute to 1L with dH20 and filter (0.22 uM).
- Assay Diluent: 5X concentrated
- Detection enzyme: pre-titrated Avidin-HRP
- Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
- Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
- 96 Well Plate: Corning Costar 9018 (included with product Cat. #’s ending in suffixes -22, -44, -76, -86)
Applications Reported
For research use only, not for diagnostic or therapeutic use.
References
Miyazono, K., et al. 1992. Structure, function and possible clinical application of transforming growth factor-beta. J Dermatol. 19: 644-647.
Roberts, A., et al. 1993. Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors 8: 1-9.
Peng, Y., et al. 2004. TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. PNAS. 101: 4572-4577.
Fantini, M., et al. 2005. TGF-beta – induced Foxp3+ regulatory T cells suppress Th1-mediated experimental colitis. Gut. [Epub ahead of print]
Zheng, S.G., et al. 2002. Generation ex vivo of TGF-b-producing regulatory T cells from CD4+CD25- precursors. J. Immunol. 169: 4183-4189.
Other Materials Needed
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Solutions for activating samples (not needed for standards)
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Buffers
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Wash Buffer: 1 x PBS, 0.05% Tween-20
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Stop Solution: 1M H3PO4 or 2N H2SO4
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Pipettes and pipettors
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Refrigerator
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96-well plate (Corning Costar 9018 or NUNC Maxisorp flat-bottom)
IMPORTANT NOTE: The use of ELISA plates which are not high affinity protein binding plates will result in suboptimal performance, e.g., low signal or inconsistent data. Do not use tissue culture plates or low protein absorption plates. Use only the Corning Costar 9018 or NUNC Maxisorp 96 well plates provided or suggested.
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96-well ELISA plate reader (microplate spectrophotometer)
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ELISA plate washer
Stability
This ELISA set is guaranteed to perform as specified at least 12 months from date of receipt if stored and handled as instructed according to this datasheet and the Certificate of Analysis, which is included with the reagents.
Storage Instructions for Cytokine Standards
The frozen cytokine standard is already aliquoted at 20 µl per vial. Upon receipt, frozen cytokine standard should be immediately stored at -80°C; stable for at least 12 months. After thawing, quick-spin vial prior to opening. Do not re-aliquot into smaller fractions. These are single use vials. Use one time and discard. For dilution of the standard, please see instructions on the Certificate of Analysis and follow these as written.
Storage Instructions for Other Set Reagents
Store at 4°C.
IMPORTANT NOTE: Be certain that no sodium azide is present in the solutions used in this assay, as this inhibits HRP enzyme activity.
Time Requirements
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1 overnight incubation
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4½-hour incubations
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1 hour washing and analyzing samples
Experimental Procedure
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Coat Corning Costar 9018 or NUNC Maxisorp 96 well ELISA plate with 100 µl/well of capture antibody in Coating Buffer* (dilute as noted on Certificate of Analysis, which is included with the reagent set). Seal the plate and incubate overnight at 4°C.
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Aspirate wells and wash 5 times with >250 µl/well Wash Buffer (diluted to 1X). Allowing time for soaking (~ 1 minute) during each wash step increases the effectiveness of the washes. Blot plate on absorbent paper to remove any residual buffer.
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Dilute 1 part 5X concentrated Assay Diluent with 4 parts DI water.* Block wells with 200 µl/well of 1X Assay Diluent. Incubate at room temperature for 1 hour. Aspirate/wash as in step 2.
*Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
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Aspirate/wash as in step 2.
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Acid Activation of Samples: To activate latent TGF-ß1 to the immunoreactive form, the samples (but not standards) must be acidified, and then neutralized. Animal serum used in culture media may contain high levels of latent TGF-ß1, so controls should be run to determine baseline concentrations of TGF-ß1 in culture media.
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Tissue culture supernatants: Per 100 ul of sample, add 20 ul of 1N HCl; incubate 10 minutes at room temperature, then neutralize with 20 ul of 1N NaOH. [When calculating final sample concentration, correct to the dilution factor of 1.4.]
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Serum or plasma: Dilute 1:5 in PBS, then treat as above for supernatants.
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Using Assay Diluent, dilute standards as noted on the Certificate of Analysis (C of A). Add 100 µl/well of standard to the appropriate wells. Perform 2-fold serial dilutions of the top standards to make the standard curve. Add 100 µl/well of your acid-activated samples to the appropriate wells. Cover or seal the plate and incubate at room temperature for 2 hours (or overnight at 4°C for maximal sensitivity).
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Aspirate/wash as in step 2. Repeat for a total of 5 washes.
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Add 100 µl/well of detection antibody diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 1 hour.
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Aspirate/wash as in step 2. Repeat for a total of 5 washes.
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Add 100 µl/well of Avidin-HRP* diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 30 minutes.
*Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
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Aspirate and wash as in step 2. In this wash step, soak wells in Wash Buffer for 1 to 2 minutes prior to aspiration. Repeat for a total of 7 washes.
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Add 100 µl/well of Substrate Solution to each well. Incubate plate at room temperature for 15 minutes.
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Add 50 µl of Stop Solution to each well.
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Read plate at 450 nm. If wavelength subtraction is available, subtract the values of 570 nm from those of 450 nm and analyze data.
Ready-SET-Go Cytokine ELISA Set Buffers:
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Assay Diluent (5X concentrate): Dilute 1/5 in DI water.
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Substrate Solution: Ready to use (1X); 100 µl per well.
Standard Calibration
The standard of the Ready-SET-Go! is calibrated against NIBSC standards:
Table 1: Table of Standard Calibration
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Table of Standard Calibration
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| Cytokine |
ng of eB standard |
ng of NIBSC standard |
U of NIBSC standard |
NIBSC Lot # |
| hIL-2
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1
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1.1
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14.6
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86/564
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| hIL-4
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1
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2.2
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22
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88/656
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| hIL-5
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1
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2.2
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22
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90/586
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| hIL-6
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1
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1.7
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170
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89/548
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| hIL-10
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1
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0.8
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4
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93/722
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| hIL-12
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1
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0.8
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8
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95/544
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| hIFN-g
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1
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1.1
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22
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87/586
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| hTNF-a
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1
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0.9
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36
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87/650
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| mIL-2
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1
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3.1
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310
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93/566
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| mIL-4
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1
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3
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30
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91/656
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| mIL-6
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1
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8.5
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850
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93/730
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| mIFN-g*
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1
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4.5
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Gg02-901-533
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| mTNF-a
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1
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1.7
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340
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88/532
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| * Mouse IFN-g is calibrated using NIH standard (Lot Gg02-901-533) and is measured in Units (U) |
Table 2: ELISA Troubleshooting Guide
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ELISA Troubleshooting Guide
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| Problem |
Possibility |
Solution |
| A. High Background
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1. Improper and inefficient washing
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1. Improve efficiency of washing. Fill plates completely, soak for 1 minute per wash, as directed
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2. Cross contamination from other specimens or positive control
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2. Repeat ELISA, be careful when washing and pipetting
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3. Contaminated substrate
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3. Substrate should be colorless
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4. Incorrect dilutions, e.g., conjugate concentration was too high
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4. Repeat test using correct dilutions; check with the recommendations of the antibody manufacturer
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| B. No signal
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1. Improper, low protein binding capacity plates were used
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1. Repeat ELISA, using recommended high binding capacity plates
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2. Wrong substrate was used
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2. Repeat ELISA, use the correct substrate
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3. Enzyme inhibitor present in buffers; e.g., sodium azide in the washing buffer and Assay Diluent inhibits peroxidase activity
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3. Repeat ELISA, make sure your system contains no enzyme inhibitor
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| C. Very weak signal
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1. Improper and inefficient washing
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1. Make sure washing procedure is done correctly
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2. Incorrect dilutions of standard
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2. Follow recommendations of standard handling exactly as written on the certificate of analysis
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3. Insufficient incubation time
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3. Repeat ELISA, follow the protocol carefully for each step’s incubation time
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4. Incorrect storage of reagents
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4. Store reagents in the correct temperature, avoid freeze and thaw, avoid using the “frost free” freezer
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5. Wrong filter in ELISA reader was used
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5. Use the correct wavelength setting
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6. Wrong plate used
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6. Use the recommended Corning Costar 9018 or NUNC Maxisorp flat bottom 96 well plates
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| D. Variation amongst replicates
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1. Improper and inefficient washing
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1. Make sure washing procedure is done correctly; see certificate of analysis
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2. Poor mixing of samples
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2. Mix samples and reagents gently and equilibrate to proper temperature
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3. Plates not clean
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3. Plates should be wiped on bottom before measuring absorbance
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4. Improper, low binding capacity plates were used
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4. Use recommended high binding capacity plates
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5. Reagents have expired
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5. Do not use if past expiration date
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