Human IL1 beta Assay Kit

IL1β, also known as leukocytic pyrogen or mononuclear cell factor, is a pro-inflammatory cytokine of the IL1 family.
IL1β 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.
Cisbio's Human IL1β kit is a homogeneous, add and read assay to measure IL1β secretion in supernatant. It offers a fast, efficient alternative to ELISA.

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 hIL1β value (pg/mL)
Species Human only
Download Insider tips for PBMC isolation Technical Note
Download cytokine release from fresh blood samples application notes

Assay principle

The assay protocol, using a Cisbio 96-well low volume or a 384-well small volume white plate (20 µL final), is described below. Cell supernatant, sample or standard are dispensed directly into the assay plate for the detection by HTRF® reagents. 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 in 96 to 1536-well plates by simply resizing each addition volume proportionally.

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.

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Cytokines assay principle add

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Cytokines assay principle read

(1) Assay data analysis:

  • "Guidelines for data reduction" button will show you how dealing with HTRF data processing and 4PL 1/y2 fitting.
  • "MyAssays" button will direct you to a free online software able to run IL1β analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

Intra-assay (n=24)

Sample Mean [IL1β] (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 [IL1β] (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.

Accuracy

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.

Assay validation

1. IL1β secretion in THP1 cells stimulated with LPS & PMA: cell densities

THP1 cells plated @ 50 and 100 kcells/well were stimulated for 18h with PMA + LPS respectively used at 50 ng/mL and 1 µg/mL. 16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL1β Assay Kit.

2. IL1β secretion in PBMCs stimulated with LPS: cell density

BPMCs plated @ 50, 100, 200 and 400 kcells/well were stimulated for 18h with various amounts of LPS ranging from 0.02 to 2 µg/mL. 16 µL of supernatants were then transferred into a white detection plate to be analyzed by the Human IL1β Assay Kit.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human IL1 beta kit - 500 tests62HIL1BPEG
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Human IL1 beta kit - 10,000 tests62HIL1BPEH
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Human IL1 beta kit - 1 x 96 tests62HIL1BPET
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Companion products

DescriptionCat. noProduct insertMSDS
Human IL1 beta kit - 3 Standards62HIL1BCD3
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Human IL1 beta kit - Standard62HIL1BCDA
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