Human IL6 kit
No-wash kit to quantify released Human IL6
Human IL1 beta kit HTRF®
The HTRF human IL beta kit is designed for the quantification of human IL1 beta release in cell supernatant.
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No-wash
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Low sample consumption
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Under 2h bench time
Overview
IL1 beta, also known as leukocytic pyrogen or mononuclear cell factor, is a pro-inflammatory cytokine of the IL1 family. IL1 beta is considered to be a major endogenous pyrogen and induces prostaglandin synthesis, T cell activation, B cell activation, and subsequent antibody production. It is also a known promoter of T cell differentiation into Th17.
Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable the detection of low levels of analytes in serum or plasma. Check out the Human IL-1β AlphaLISA kit’s analytical performances for more information or learn more about AlphaLISA. When assaying, always follow the recommended protocol and avoid highly haemolyzed samples.
Benefits
- VALIDATED ON THP1 AND PBMC
- PANEL QUANTIFICATION
Assay principle
Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Cisbio 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.
To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page.
Cisbio also worked with Myassays.com to help you in your data analysis. By clicking on this link, you’ll be able to access a free online software to run your IL1 beta analysis.
Technical specifications of human IL1 beta kit
| Sample size | 16 µL |
| Final assay volume | 20 µL |
| Kit components | Lyophilized standard, frozen detection antibodies, buffers &protocol. |
| LOD &LOQ (in Diluent) | 5 pg/mL &39.4 pg/mL |
| Range | 39.4 6,500 pg/mL |
| Time to result | Overnight at RT |
| Calibration | NIBSC (86/680) value (IU/mL) = 0,1 x HTRF hIL1ß value (pg/mL) |
| Species | Human only |
Intra and inter assay
Intra-assay (n=24)
| Sample | Mean [IL1ß] (pg/mL) | CV |
|---|---|---|
| 1 | 65 | 18% |
| 2 | 705 | 9% |
| 3 | 5030 | 4% |
| Mean CV | 10% |
Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.
Inter-assay (n=4)
| Sample | [IL1ß] (pg/mL) | Mean (delta R) | CV |
|---|---|---|---|
| 1 | 159 | 378 | 15% |
| 2 | 702 | 1742 | 13% |
| 3 | 3095 | 7736 | 12% |
| Mean CV | 13% |
Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.
Dilutional linearity
Sample | Dilution factor | [IL1β] expected (pg/mL) | [IL1β] detected (pg/mL) | Recovery |
| 1 | 1 | - | 5486 | - |
| 4.3 | 1276 | 1202 | 94% | |
| 10.7 | 513 | 479 | 93% | |
| Mean | 93% | |||
| 2 | 1 | - | - | - |
| 4.3 | 408 | 378 | 93% | |
| 8.9 | 197 | 194 | 99% | |
| 18.5 | 95 | 95 | 98% | |
| Mean | 97% | |||
The excellent % of recovery obtained from these experiments show the good linearity of the assay.
Spike and recovery
Sample | [IL1β] added (pg/mL) | [IL1β] expected (pg/mL) | [IL1β] detected (pg/mL) | Recovery |
| 1 | 3250 | 5057 | 5323 | 105% |
| 2 | 3250 | 7326 | 7488 | 102% |
| Mean Recovery | 104% |
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.
IL1ß secretion in THP1 cells stimulated with LPS & PMA
THP1 cells plated at 50 and 100 kcells/well were stimulated for 18 h with PMA and LPS respectively used at 50 ng/mL and 1 µg/mL. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed with the Human IL1ß Assay.
IL1ß secretion in PBMC stimulated with LPS
PBMC plated at 50, 100, 200 and 400 kcells/well were stimulated for 18 h with increasing concentrations of LPS ranging from 0.02 to 2 µg/mL. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human IL1ß Assay Kit.
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Plate Reader Requirement
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