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HTRF High Performance Human IL-6 Detection Kit, 96 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 and disposal requirements under European REACH regulations (EC 1907/2006).

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Product Variants
Part number: 62HIL6V2PEG
Unit Size: 500 Assay Points
List price: USD 1,103.00
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USD 0.00
USD 1,103.00 /each
Part number: 62HIL6V2PEH
Unit Size: 10,000 Assay Points
List price: USD 10,290.00
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USD 0.00
USD 10,290.00 /each
Part number: 62HIL6V2PET
Unit Size: 96 Assay Points
List price: USD 652.00
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USD 652.00
USD 652.00 /each

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

Application
Protein Quantification
Assay Target
IL-6
Assay Target Class
Cytokines
Assay Technology
TR-FRET
Brand
HTRF
Detection Method
HTRF
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Inflammation
Neuroscience
Oncology
Unit Size
96 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 principle
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

assay details
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
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.

assay validation
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.

assay validation
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

assay validation

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