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HTRF Human IL-12/IL-23 p40 Detection Kit, 500 Assay Points

The HTRF Human IL12/23 p40 kit is designed for the quantification of human IL12/23 p40 release in cell supernatant. The assay is compatible with human samples only, and is highly specific for p40, cross-reacting with all p40-containing cytokines such as IL12, IL23, p40 monomer, and p40-p40 (p80) dimer.

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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Feature Specification
Application Protein Quantification
Dynamic Range 71 - 10,000 pg/mL
Limit of Detection 15 pg/mL
Limit of Quantification 71 pg/mL
Sample Volume 16 µL

The HTRF Human IL12/23 p40 kit is designed for the quantification of human IL12/23 p40 release in cell supernatant. The assay is compatible with human samples only, and is highly specific for p40, cross-reacting with all p40-containing cytokines such as IL12, IL23, p40 monomer, and p40-p40 (p80) dimer.

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).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 500 Assay Points
Part #:
62HIL12P40PEG
List Price
USD 1,103.00
Unit Size: 10,000 Assay Points
Part #:
62HIL12P40PEH
List Price
USD 10,290.00

Overview

p40 is a common component of the p40 family of cytokines which encompasses IL-12 (p40+p35 heterodimer), IL-23 (p40+p19 heterodimer), p40 (monomer), and p80 homodimer (p40+p40). Detection of the shared subunit p40 allows detection of all of these 4 members. These cytokines are instrumental in inflammation and immuno-regulation.

Specifications

Application
Protein Quantification
Brand
HTRF
Detection Modality
HTRF
Dynamic Range
71 - 10,000 pg/mL
Limit of Detection
15 pg/mL
Limit of Quantification
71 pg/mL
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target Class
Cytokines
Target Species
Human
Technology
TR-FRET
Unit Size
500 Assay Points

Video gallery

How it works

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 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 in up to a 1536-well format by simply resizing each addition volume proportionally.

 

cytokines-how-it-works-assay-protocol
Assay principle - p40 family

The assay is compatible with human samples only, and is highly specific for p40, cross-reacting with all p40-containing cytokines such as IL12, IL23, p40 monomer, and p40-p40 (p80) dimer.

cytokines-how-it-works-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. To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page. Revvity also works 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 IL12/23 p40 analysis.

 

Assay details

Standard curve

The IL12/23 p40 standard curve is generated in the assay Diluent 5 provided in the kit.

specification-il12-il23-p40-standard
Assay specifications
Sample size 16 µL
Final assay volume 20 µL
Kit components Lyophilized standard, frozen detection antibodies, buffers & protocol
LOD & LOQ (in Diluent) 15 pg/mL & 71 pg/mL
LOD & LOQ (in DMEM+10%FCS) 56 & 96 pg/mL
LOD & LOQ (in RPMI+10%FCS) 55 & 109 pg/mL
Range 71 – 10 000 pg/mL
Time to result Overnight at RT
Correspondance to I.S. NIBSC (95/544) IL12 value (IU/mL) = 0,01 x HTRF hIL12/23 p40 value (pg/mL)
Species Human only

 

Analytical performance

Intra and inter assay

Intra-assay (n=24)

Sample Mean [IL12/23-p40] (pg/mL) CV
1 218 9.4%
2 4797 7.6%
3 8499 8.3%
  Mean CV 8.4%

 

Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

Samples were cell culture supernatants from stimulated THP1 cells.

Inter-assay (n=3 experiments)

Sample Mean [IL12/23-p40] (pg/mL) CV
1 191 7.6%
2 4739 1.7%
3 8138 4.6%
  Mean CV 4.6%

 

Each of the samples was measured in 3 independent experiments (3 days), and % CV was calculated for each sample. Samples were cell culture supernatants from stimulated THP1 cells.

Dilutional linearity
Dilution Factor [IL12/23-p40] Expected ( pg / mL ) [IL12/23-p40] Measured ( pg / mL ) Dilution Recovery
Neat - 6073.4 100%
2 3036.7 2837.7 91%
4 1518.3 1379.1 91%
8 759.2 610.3 80%
16 379.6 310.7 82%
32 189.8 169.0 89%
  Summary   89%

 

The excellent % of recovery obtained from these experiments show the good dilution linearity of the assay. Samples were THP1 cell supernatants serially diluted with the DIL5 kit diluent.

Antigen spike recovery
Sample

[IL12/23-p40] Standard

(pg/mL)

[IL12/23-p40]

Spiked Sample (pg/mL)

Expected (pg/mL) Measured (pg/mL) Recovery
1 2886 6148 9034 9444 105%
2805 5691 5885 103%
1365 4251 4529 107%
2 1410 2732 4142 4353 105%
1312 2723 2908 107%
661 2071 2126 103%
3 98 272 370 413 112%
111 209 259 124%
45 143 184 129%
Mean CV       111%


​THP1 cell supernatant samples were spiked to 3 levels of recombinant p40, and the response measured was compared to the calculated expected values. The 100% of recovery indicates similar measurements of cytokine from samples and the kit standard.

Cross-reactivities

Tested Protein
Cross Reactivity
Human p40 monomer 100%
Human p40 homodimer (p40+p40 = p80) 65%
Human IL12 (p40+p35 = p70) 62%
Human IL23 (p40+p19) 51%
Mouse p40 homodimer (p40+p40 = p80) 0%
Mouse IL12 0%
Mouse IL23 0%
Rat IL12 0%
Rat IL23 0%

 

Cross reactivities were assessed using recombinant proteins from the p40 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 measure concentrations. Cross-reactivity was calculated as a mean of 6 tested concentrations from 116 to 10.000 pg/mL. The assay is human specific, as mouse and rat p40 cytokines were not detected using this assay.

Assay validation

IL12/23 p40 secretion from LPS-stimulated THP1 human monocytes

The human monocyte cell line THP1 was used for an IL12/23-p40 secretion study using LPS as an agonist. THP1 cells were cultured in 10% FCS supplemented RPMI1640 medium at 37°C, 5% CO2. 50 µL of THP1 cells in suspension were transferred into a 96-well cell-culture plate at densities of 200 and 400 k cells/well. Cells were stimulated for 24h with increasing doses of LPS in a final volume of 100 µL.  16 µL of supernatants were then transferred into a white detection plate (384 low volume), and 4 µL of the HTRF Human IL12/23-p40 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. As expected, increasing doses of LPS led to incremental releases of IL12/23-p40 cytokines. The concentrations measured were lower using the highest density, indicating that the 200k density was the best compromise to study the secretion of the cytokines using this cell model in suspension.

assay-validation

 

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