HTRF Human IL12/23 p40 Detection Kit HTRF®

Associated products

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.

Low sample consumption
No-wash
Enables fast assay qualification

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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.

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 PerkinElmer 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.

Principle of the HTRF Human IL12/23 p40 assay

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.

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. PerkinElmer 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.

Standard curve

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

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

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.

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 kcells/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.

Validation of HTRF Human IL12/23-p40 kit on THP1 cell line

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Product Insert IL12/23 p40 Kit / 62HIL12P40PEG-62HIL12P40PEH

62HIL12P40PEG-62HIL12P40PEH - Product Insert

Safety Data Sheet (DEU) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (ELL) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (FRA-FR) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (ITA) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (SPA) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (ENG-GB) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (ENG-US) IL12/23 p40 Kit / 62HIL12P40PEG

62HIL12P40PEG - Safety Data Sheet

Safety Data Sheet (DEU) IL12/23 p40 Kit / 62HIL12P40PEH