HTRF Human CCL22 Detection kit HTRF®

Name: HTRF Human CCL22 Detection kit
Brand: Cisbio
SKU: 46139
Availability: InStock

Associated products

The HTRF Human CCL22 kit is designed for the quantification of human CCL22 release in cell supernatant.

Low sample consumption
No-wash
Faster and more convenient than ELISA

Download the protocols

The HTRF Human CCL22 kit is designed for the quantification of human CCL22 release in cell supernatant.

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Overview

CCL22 (C-C motif chemokine 22), previously referred to as Macrophage-derived chemokine (MDC), is a chemokine secreted by dendritic cells (DC), macrophages, and activated B-lymphocytes. CCL22 binds to CCR4 present in many different cell types.

CCL22 is secreted by M2 macrophages, which contribute to Th2 responses such as phagocytosis, tissue repair, and wound healing.

Through its binding to CCR4, CCL22 has also been shown to contribute to the pathogenesis of atopic diseases, like asthma and atopic dermatitis (AD).

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 CCL22 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 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 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.

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 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 CCL22 analysis.

Technical specifications of human CCL22 kit

Sample size	16 µL
Final assay volume	20 µL
Kit components	Lyophilized standard, frozen detection antibodies, buffers & protocol.
LOD &LOQ (in Diluent)	5.7 pg/mL & 7.9 pg/mL
LOD &LOQ (in RPMI)	9.8 pg/mL & 16.8 pg/mL
LOD &LOQ (in DMEM)	7.7 pg/mL & 13.8 pg/mL
Range	35 – 4,000 pg/mL
Time to result	4h to Overnight at RT
Species	Human only

Intra and inter assay

Intra-assay (n=24)

Sample	Mean [CCL22] (pg/mL)	CV
1	584	9%
2	284	4%
3	139	7%
	Mean CV	6%

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

Inter-assay (n=4)

Sample	Mean [CCL22] (pg/mL)	CV
1	560	7%
2	396	9%
	Mean CV	9%

Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.

Dilutional linearity

Sample	Dilution factor	[CCL22] expected (pg/mL)	[CCL22] detected (pg/mL)	Recovery
1	1	-	584	-
	2	292	284	97%
	4	142	139	98%
	Mean			97%
2	1	-	1168	-
	2	584	584	100%
	4	292	262	90%
	Mean			95%

The excellent % of recovery obtained from these experiments show the good linearity of the assay.

Spike recovery

Different amounts of recombinant cytokine (P1, P2 or P3) were added to 2 different PBMC supernatant stimulated samples (S1 and S2).

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.

RPMI	C recomb prot (pg/ml)	C expected (pg/ml)	C obtained (pg/ml)	% Recovery
S1 Native	0	_	596	_
S1-P1 spike	845	1440	1520	95%
S1-P2 spike	401	997	1040	96%
S1-P3 spike	167	763	817	93%
mean				95%
S2 Native	0	_	350	_
S2-P1 spike	845	1194	1096	109%
S2-P2 spike	401	751	734	102%
Mean				105%

CCL22 secretion in PBMC cells stimulated with PHA-P and PHA-M

PBMC cells plated at 200 and 300 kcells/well were stimulated for 72 h with PHA-P and PHA-M respectively, used at 10 µg/mL.

16 µL of supernatants were then transferred into a white detection plate (384 low volume) and 4 µL of the HTRF Human CCL22 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

The standard curve prepared in RPMI allowed the estimation of the CCL22 concentration in each sample. The results showed that PHA-M stimulated more CCL22 secretion than PHA-P. Little CCL22 was detected in the non-stimulated sample.

CCL22 estimation with HTRF human CCL22 in PBMC cells stimulated or not with PHA-P and PHA-M

Calibration with Other Technologies

Different densities of PBMC cells were stimulated for 48 or 72 h with 10 µg/mL of PHA-P. The CCL22 concentration of the different samples (pure and diluted) was estimated using the HTRF Human CCL22 detection kit, the AlphaLISA human CCL22 detection, and an ELISA assay.

For the HTRF assay, 16 µL of the different cell supernatants were then transferred into a white detection plate (384 low volume) and 4 µL of the HTRF Human CCL22 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

For the AlphaLISA assay, 5 µL of cell supernatants were transferred into a grey detection plate (384w plate) and 45µL of detection reagents were added. The AlphaLISA counts were recorded after a 3h incubation at room temperature.

The AlphaLISA human CCL22 detection assay and the HTRF human CCL22 detection kit gave the same concentration estimation for each sample, while the ELISA assay estimated concentration values only half those estimated by the HTRF and AlphaLISA assays.

Cytokine assays: a guide to success with HTRF

Setting up a successful cytokine assay - Guides

Portfolio of Life Sciences Cytokine Assays

This leaflet presents the analytical performances of each assay - Brochures

Product Insert CCL22 (h) Kit / 62HCCL22PEG-62HCCL22PEH

62HCCL22PEG-62HCCL22PEH - Product Insert

Safety Data Sheet (ELL) CCL22 (h) Kit / 62HCCL22PEG