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HTRF High Performance Human IL-6 Detection Kit,500 Assay Points
The HTRF High Performance Human IL-6 kit is designed for the simple and rapid quantification human IL-6 release from cell supernatants. This kit is the new and improved version of part number 62HIL06PE, the Human IL-6 Detection Kit.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Product information
Overview
IL-6 is a pro-inflammatory cytokine involved in acute-phase reaction, inflammation, and cancer progression. Secreted by T cells, macrophages, and fibroblasts, it induces B and T cell proliferation. It is being studied in a wide variety of research areas including diabetes, Alzheimer's disease, depression, and several cancers. Along with TGF beta, IL-6 is main promoter of T cell differentiation into TH17, a new component of immuno-oncology.
Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable detection of low levels of analytes in serum or plasma.
Specifications
| Assay Target Class |
Cytokine
|
|---|---|
| Brand |
HTRF
|
| Detection Method |
HTRF
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Therapeutic Area |
Inflammation
Neuroscience
Oncology
|
| Unit Size |
500 Assay Points
|
How it works
Principle of the HTRF HP human IL-6 assay
Cell supernatant sample, or standard is dispensed directly into the assay plate for the detection by HTRFTM reagents (384-well low-volume white plate or Revvity low-volume 96-well plate in 20 µl). The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run up to a 1536-well format by simply resizing each addition volume proportionally.
Assay data analysis
The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases.
Assay details
HTRF HP human IL-6 standard curve
The HP human IL-6 standard curve is generated in the assay Diluent 5 provided in the kit
| Sample size | 16 µL |
|---|---|
| Final assay volume | 20 µL |
| Kit components | Lyophilized standard, frozen detection antibodies, buffers & protocol |
| LOD & LOQ (in Diluent) | 10 pg/mL & 16 pg/mL |
| LOD & LOQ (in DMEM+10%FCS) | 16 & 24 pg/mL |
| LOD & LOQ (in RPMI+10%FCS) | 11 & 21 pg/mL |
| Range | 38.8 - 12000 pg/mL |
| Time to result | 2h at RT |
| Correspondance to I.S. | NIBSC (89/548) value (IU/mL) = 0,1 x HTRF HP hIL6 value (pg/mL) |
| Species | Human only |
Analytical performance
Intra-assay precision table
Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.
| Sample | Mean [IL-6](pg/mL) | CV |
|---|---|---|
| 1 | 47 | 14% |
| 2 | 695 | 7% |
| 3 | 4588 | 5% |
| Mean CV | 9% |
Inter-assay precision table
Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample.
| Sample | Mean [IL-6] (pg/mL) | CV |
|---|---|---|
| 1 | 96 | 10% |
| 2 | 1862 | 6% |
| 3 | 4829 | 5% |
| Mean CV | 7% |
Dilutional linearity
The recovery percentages obtained from these experiments show the good linearity of the assay.
| Dilution Range | Dilution factor | [IL-6] Expected (pg/mL) | [IL-6] detected (pg/mL) | Recovery |
|---|---|---|---|---|
| 1 | 1 | 10254 | 9841 | 96% |
| 2 | 4920,5 | 4776 | 97% | |
| 4 | 2460 | 2338 | 95% | |
| 8 | 1230 | 1080 | 88% | |
| 16,5 | 615,1 | 501 | 82% | |
| Mean | 91% | |||
| 2 | 1 | 10254 | 10465 | 102% |
| 1,5 | 6976 | 6763 | 97% | |
| 2,25 | 4651 | 4510 | 97% | |
| 3,37 | 3100 | 2761 | 89% | |
| 5,07 | 2067 | 1793 | 87% | |
| Mean | 94% | |||
Spike and recovery
The same amount of recombinant cytokine was added to 2 different serum samples, and the set of responses obtained from a standard curve was compared to the calculated expected values. The ~ 100% of recovery observed validates the sample matrix used for this assay.
| Sample |
[IL-6] Spiked Sample (pg/mL) |
[IL-6] Standard (pg/mL) |
Expected (pg/mL) |
Measured (pg/mL) |
Recovery |
|---|---|---|---|---|---|
| 1 | 1855 | 210 | 1033 | 947 | 92% |
| 1406 | 1645.5 | 1512 | 92% | ||
| 2 |
10254 |
210 | 5232 | 4758 | 91% |
| 1406 | 5830 | 6013; | 103% | ||
| Mean CV | 94% |
Cross reactivities
Cross reactivities were assessed using recombinant proteins from the IL-6 cytokine family. Proteins were tested up to 10 000 pg/mL and standard curves were generated for each. Signals were interpolated on the assay standard curve to interpolate concentrations.
| Tested protein | Cross reactivity |
|---|---|
| Human IL-6 | 100% |
| Mouse IL-6 | 0% |
| Cynomolgus IL-6 | 3% |
Assay validation
hIL-6 secretion in PBMCs cells stimulated with LPS
PBMC plated at 50, 100, 200 and 400 kcells/well were stimulated for 18h with LPS at 0 to 2 µg/mL. Then 16 µL of supernatants pre-diluted were transferred into a white detection plate (384 low volume) to be analyzed using the HP Human IL-6 Assay Kit.
Effect of dexamethasone on PBMC induced IL-6 release
PBMCs plated at 200 kcells/well were stimulated for 18h with increasing concentrations of dexamethasone in presence of 2 µg/mL LPS. Then 16 µL of supernatants pre-diluted were transferred into a white detection plate (384 low volume) to be analyzed using the HP Human IL-6 Assay Kit.
Effect of JTE-607 on PBMC induced IL-6 release
PBMCs plated 50, 100, 200 and 400 kcells/well were stimulated for 18h with 5 µM of JTE-607 in presence of 0,2 µg/mL LPS. Then 16 µL of supernatants pre-diluted were transferred into a white detection plate (384 low volume) to be analyzed using the HP Human IL-6 Assay Kit
Resources
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Brochure
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SDS, COAs, Manuals and more
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