TGF-beta 3 Magnetic Luminex® Performance Assay
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TGF-beta 3 Magnetic Luminex® Performance Assay Summary
| Panel | Luminex Performance Human TGF-beta Panel |
| Assay Type | Magnetic bead-based multiplex assay for the Luminex® platform |
| Format | 1 x 96-well microplate and Magnetic antibody-coated beads |
| Species | Human, Mouse, Rat, Porcine |
| Analytes Detected | Please see analyte list in assay customization tool below. |
| Performance Validation | Our Luminex High Performance Assays undergo our most extensive validation testing. All Luminex High Performance Assay Panels are validated for use with serum and plasma samples. Some High Performance Assays are additionally validated for cell culture supernatants, milk, saliva, or urine, as indicated on the individual product data sheets. Luminex High Performance Assays are tested for sensitivity (three-quarters the low standard), intra-assay precision, inter-assay precision, and to ensure assay linearity for validated sample types. Recovery values for individual samples in validated sample types are also tested. Validation data for each analyte can be found in the product datasheet. |
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Magnetic Luminex High Performance Assays are flexible bead-based multiplex assays. They allow up to 50 user-defined target analytes to be simultaneously profiled using cell culture supernates, serum, or plasma samples. Some panels are also validated with human milk, saliva, and/or urine samples.
- Simultaneously profile Human TGF-beta 3 with up to analytes of your choice in one sample
- Magnetic format allows for easier wash steps
- Undergoes similar validation testing as Quantikine® ELISA Kits
- Precise and Specific
- Requires a small sample volume (<50 µL)
- Run your assay in just 3-5 hours
- Mass-calibrated standards for consistent results with every new lot of material
- Enough reagents for one 96-well plate
- Standard Cocktail(s)
- Bead Diluent
- Biotin Antibody Diluent
- Calibrator Diluent
- Wash Buffer
- Streptavidin-PE
- One flat-bottom 96-well Microplate
- Foil Plate Sealers (4)
- Mixing Bottle
- Standard Value Card**
**A standard curve must be generated each time an assay is run, utilizing values from the Standard Value Card included in the Base Kit.
- User-mix antibody-coated Magnetic beads in individual vials
- User-mixed cocktail of biotinylated detection antibody
Precision
|
Intra-assay Precision (precision within an assay) - Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. |
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| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 94 | 94 | 94 |
| Mean (pg/mL) | 174 | 1499 | 11,266 | 158 | 1498 | 10,672 |
| Standard deviation | 17 | 137 | 672 | 27 | 186 | 1208 |
| CV (%) | 9.8 | 9.1 | 6.0 | 17.1 | 12.4 | 11.3 |
Recovery
|
Samples containing and/or spiked with human TGF-B1 were evaluated for recovery. |
||||||
| Sample Type | Average % Recovery | Range (%) | ||||
|---|---|---|---|---|---|---|
| Cell culture supernate | 96 | 65-124 | ||||
| Serum | 76 | 65-92 | ||||
| EDTA plasma | 82 | 73-97 | ||||
| Heparin plasma | 83 | 72-98 | ||||
| Platelet-poor EDTA plasma | 89 | 69-110 | ||||
| Platelet-poor heparin plasma | 88 | 64-115 | ||||
| Urine | 106 | 86-125 | ||||
Linearity
|
Samples containing and/or spiked with human TGF-B1 were serially diluted to evaluate assay linearity. |
||||||||||||
| Cell culture supernate | Serum | EDTA plasma | Heparin plasma | Platelet-poor EDTA plasma | Platelet-poor Heparin plasma | Urine | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1:2 | Average % of Expected | 98 | 103 | 100 | 100 | 106 | 108 | 93 | ||||
| Range (%) | 83-120 | 96-113 | 81-111 | 90-112 | 94-120 | 90-123 | 82-109 | |||||
| 1:4 | Average % of Expected | 91 | 108 | 101 | 102 | 103 | 103 | 83 | ||||
| Range (%) | 75-120 | 97-122 | 83-114 | 91-119 | 83-127 | 84-125 | 69-99 | |||||
| 1:8 | Average % of Expected | 88 | 109 | 101 | 101 | 104 | 103 | 80 | ||||
| Range (%) | 72-114 | 96-119 | 83-111 | 86-111 | 82-129 | 80-130 | 66-107 | |||||
Assays for the Luminex platform are offered as High Performance Assays or Assays. The Luminex High Performance Assays are fully validated panels with a focused selection of analytes. They are available in We Mix, You Mix, and Predetermined formats. The Luminex Assays allows for the maximum number of analytes in a multiplex and is supplied as a premixed kit. View a table comparing the features and benefits of these bead-based multiplex assays.
Magnetic Luminex High Performance Assays are designed for use with the Luminex MAGPIX CCD Imager. Alternatively, kits can be used with the Luminex 100/200™ or FLEXMAP 3D, dual laser, flow-based sorting and detection platforms.
Analyte-specific antibodies are pre-coated onto color-coded microparticles. Microparticles, standards, and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest. After washing away any unbound substances, a biotinylated antibody cocktail specific to the analytes of interest is added to each well. Following a wash to remove any unbound biotinylated antibody, Streptavidin-Phycoerythrin conjugate (Streptavidin-PE), which binds to the biotinylated detection antibodies, is added to each well. A final wash removes unbound Streptavidin-PE and the microparticles are resuspended in buffer and read using the Luminex or Bio-Plex analyzer. One laser is bead-specific and determines which analyte is being detected. A magnet in the analyzer captures and holds the superparaMagnetic microparticles in a monolayer. Two spectrally distinct Light Emitting Diodes (LEDs) illuminate the beads. One LED identifies the analyte that is being detected and the second LED determines the magnitude of the PE-derived signal, which is in direct proportion to the amount of analyte bound. Each well is imaged with a CCD camera. Kits can also be used with Luminex 100/200 or a Bio-Rad Bio-Plex dual laser, flow-based systems.
Scientific Data
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Quantist software graph of signal intensity versus concentration for TGF-beta 3 in the Human TGF-beta Luminex Panel
Background: TGF-beta 3
Transforming Growth Factor Beta 1, 2, and 3 (TGF-beta 1, TGF-beta 2, and TGF-beta 3) are highly pleiotropic cytokines that virtually all cell types secrete. TGF-beta molecules are proposed to act as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. Targeted deletions of these genes in mice show that each TGF-beta isoform has some non-redundant functions: TGF-beta 1 is involved in hematopoiesis and endothelial differentiation; TGF-beta 2 affects development of cardiac, lung, craniofacial, limb, eye, ear, and urogenital systems; and TGF-beta 3 influences palatogenesis and pulmonary development. The full range of in vitro biological activities of TGF-beta 5 has not yet been explored. However, TGF-beta 1, TGF-beta 2, TGF-beta 3, and TGF-beta 5 have been found to be largely interchangeable in an inhibitory bioassay, and it is anticipated that TGF-beta 5 will show a spectrum of activities similar to the other TGF-beta family members. To date, the production of TGF-beta 5 has only been demonstrated in Xenopus.
TGF-beta ligands are initially synthesized as precursor proteins that undergo proteolytic cleavage. The mature segments form active ligand dimers via a disulfide-rich core consisting of the characteristic 'cysteine knot'. TGF-beta signaling begins with binding to a complex of the accessory receptor betaglycan (also known as TGF-beta RIII) and a type II serine/threonine kinase receptor termed TGF-beta RII. This receptor then phosphorylates and activates a type I serine/threonine kinase receptor, either ALK-1 or TGF-beta RI (also called ALK-5). The activated type I receptor phosphorylates and activates Smad proteins that regulate transcription. Use of other signaling pathways that are Smad-independent allows for distinct actions observed in response to TGF-beta in different contexts.
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