Human TNF-alpha XL Luminex® Performance Assay
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Human TNF-alpha XL Luminex® Performance Assay Summary
| Panel | Luminex Performance Human XL Cytokine 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 |
| 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. View Luminex High Performance Human XL validation data. |
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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 TNF-alpha 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
- Premixed cocktail of biotinylated detection antibody
- 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**
- High and low controls with Control Mean 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
Recommended Sample Types: Cell culture supernates, serum, EDTA plasma, and heparin plasma
Components: Human TNF-α Magnetic Microparticles (Part 898806) is supplied as a 100X concentrated stock (0.075 mL) with preservatives.
Other Supplies Required: Magnetic Luminex® Performance Assay Human XL Discovery Base Kit (R&D Systems®, Catalog # LUXLM000).
Sensitivity: The Minimum Detectable Dose (MDD) was determined by adding two standard deviations to the mean MFI of twenty zero standard replicates and calculating the corresponding concentration.
Six assays were evaluated, and the MDD of human TNF-α ranged from 0.302-2.24 pg/mL. The mean MDD was 0.615 pg/mL.
Correlation: This assay has been correlated to the Quantikine® ELISA Kit.
Specificity: This assay recognizes natural and recombinant human TNF-α.
Bead Region: 74
Precision
|
Intra-assay Precision (precision within an assay) - Two samples of known concentration were tested twenty times on one plate to assess intra-assay precision. |
||||
| Intra-Assay Precision | Inter-Assay Precision | |||
|---|---|---|---|---|
| Sample | 1 | 2 | 1 | 2 |
| n | 20 | 20 | 30 | 30 |
| Mean (pg/mL) | 118 | 1111 | 119 | 1,145 |
| Standard deviation | 3.91 | 30.6 | 16.6 | 77.2 |
| CV (%) | 3.3 | 75.4 | 13.9 | 74.0 |
Recovery
|
Samples containing and/or spiked with human TNF-alpha were evaluated for recovery. |
||||||
| Sample Type | Average % Recovery | Range (%) | ||||
|---|---|---|---|---|---|---|
| Cell culture supernate | 118 | 109-127 | ||||
| Serum | 70.5 | 59.0-84.9 | ||||
| EDTA plasma | 77.0 | 63.4-88.0 | ||||
| Heparin plasma | 77.3 | 65.5-87.2 | ||||
| Plaelet-poor EDTA | 63.8 | 44.3-83.5 | ||||
| Platelet-poor Heparin | 63.0 | 42.3-81.6 | ||||
Linearity
|
Samples containing and/or spiked with human TNF-alpha were serially diluted to evaluate assay linearity. |
||||||||||||
| Cell culture supernate | Serum | EDTA plasma | Heparin plasma | Platelet-poor EDTA | Platelet-poor Heparin | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1:2 | Average % of Expected | 93.5 | 122 | 118 | 120 | 129 | 125 | |||||
| Range (%) | 89.7-99.9 | 111-135 | 107-129 | 101-131 | 117-140 | 115-135 | ||||||
| 1:4 | Average % of Expected | 89.5 | 126 | 120 | 120 | 149 | 146 | |||||
| Range (%) | 82.2-97.5 | 110-141 | 109-137 | 103-131 | 136-161 | 129-154 | ||||||
| 1:8 | Average % of Expected | 91.3 | 135 | 128 | 124 | 172 | 160 | |||||
| Range (%) | 86.3-97.1 | 116-158 | 113-147 | 103-142 | 158-191 | 128-196 | ||||||
| 1:16 | Average % of Expected | 91.7 | 135 | 129 | 128 | 178 | 163 | |||||
| Range (%) | 88.2-95.2 | 125-148 | 115-141 | 116-138 | 147-213 | 124-221 | ||||||
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
Background: TNF-alpha
Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, apoptosis, and immune system development. TNF-α is produced by a wide variety of immune and epithelial cell types. Human TNF-α consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD). Within the ECD, human TNF-α shares 97% aa sequence identity with rhesus and 71% - 92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-α. The 26 kDa type 2 transmembrane protein is assembled intracellularly to form a noncovalently linked homotrimer. Ligation of this complex induces reverse signaling that promotes lymphocyte costimulation but diminishes monocyte responsiveness.
Cleavage of membrane bound TNF-α by TACE/ADAM17 releases a 55 kDa soluble trimeric form of TNF-α. TNF-α trimers bind the ubiquitous TNF RI and the hematopoietic cell-restricted TNF RII, both of which are also expressed as homotrimers. TNF-α regulates lymphoid tissue development through control of apoptosis. It also promotes inflammatory responses by inducing the activation of vascular endothelial cells and macrophages. TNF-α is a key cytokine in the development of several inflammatory disorders. It contributes to the development of type 2 diabetes through its effects on insulin resistance and fatty acid metabolism.
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