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HTRF Human CCL17 (TARC) Detection Kit, 10,000 assay points

CCL17 Product image
HTRF Human CCL17 (TARC) Detection Kit, 10,000 assay points
CCL17 Product image
CCL17 Product image
CCL17 Product image
CCL17 Product image
CCL17 Product image

The HTRF human CCL17 kit is for the simple and rapid quantification of soluble human CCL17 proteins in cell culture supernatants, and can be used as a no-wash alternative to more traditional wash-based ELISA.

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Feature Specification
Application Protein Quantification
Dynamic Range 21-5500 pg/mL
Limit of Detection 12 pg/mL
Limit of Quantification 21 pg/mL
Sample Volume 16 µL
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The HTRF human CCL17 kit is for the simple and rapid quantification of soluble human CCL17 proteins in cell culture supernatants, and can be used as a no-wash alternative to more traditional wash-based ELISA.

View product information
Click to copy promo code to clipboard.
img-icon-10-off-white-yellow.svg

SAVE 10% on your first Revvity.com order. Use promo code below.

HELLO10

Terms and conditions apply.

Product Variants
Unit Size: 500 assay points
Part #:
62HCCL17PEG
List Price
USD 1,103.00
Your online price:
Unit Size: 10,000 assay points
Part #:
62HCCL17PEH
List Price
USD 10,290.00
Your online price:
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).
Total list price:
USD 10,290.00
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CCL17 Product image
CCL17 Product image
HTRF Human CCL17 (TARC) Detection Kit, 10,000 assay points
CCL17 Product image
CCL17 Product image
CCL17 Product image
HTRF Human CCL17 (TARC) Detection Kit, 10,000 assay points
CCL17 Product image
CCL17 Product image

Product information

Overview

Human CCL17, also known as thymus and activation regulated chemokine (TARC), was initially isolated from phytohemagglutinin-stimulated peripheral blood mononuclear cells. CCL17 is constitutively expressed in the thymus and, under activation, in several cell types. CCL17-mediated recruitment of Th2 cells and CLA+ CD4+ T cells plays a key role in allergic diseases such as atopic dermatitis, allergic asthma, allergic rhinitis, and allergic contact dermatitis. In addition, CCL17 has been detected in idiopathic pulmonary fibrosis. CCL17 and CCL22 secreted by dendritic cells (DCs) seem to mediate the recruitment of regulatory T cells to sites of inflammation in patients with chronic hepatitis.

Specifications

Application
Protein Quantification
Brand
HTRF
Detection Modality
HTRF
Dynamic Range
21-5500 pg/mL
Limit of Detection
12 pg/mL
Limit of Quantification
21 pg/mL
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
CCL17
Target Class
Cytokines
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Inflammation
Unit Size
10,000 assay points

How it works

Principle of the HTRF human CCL17 assay

The HTRF human CCL17 assay is based on a TR-FRET sandwich immunoassay involving two specific antibodies, one labelled with Eu3+-cryptate (donor) and the other with d2 (acceptor). Both antibodies are directed against the CCL17 protein. They bind to soluble CCL17, and the donor-acceptor proximity leads to a fluorescent TR-FRET signal. The intensity of the signal is directly proportional to the concentration of soluble CCL17 present in the sample (cell supernatant).

 

Assay-principle-CCL17-how-it-works-62HCCL17PEG-62HCCL17PEH-62HCCL17PEJ.jpg

 Protocol of the HTRF human CCL17 assay

The HTRF human CCL17 assay can be run in 96- or 384-well low volume white detection plates (20 µL final). As described here, samples (cell supernatants) or standards are dispensed directly into the assay plate for the detection of human CCL17 by HTRF reagents. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally. 

Assay protocol CCL17 how it works 62HCCL17PEG 62HCCL17PEH 62HCCL17PEJ.jpg

 

Assay details

Human CCL17 assay details
Sample size 16 µL
Final assay volume 20 µL
Time to result 2h at RT
Kit component Frozen detection antibodies, lyophilized standard & buffers
Species Human, some cross-reactivity with mouse protein

Analytical performance

Intra-assay precision table

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample. Samples were supernatants from PHA-treated PBMC cells.

Sample [CCL17]  (pg/mL) CV
1 1874 8.1%
2 924 7.5%
3 501 10.5%
  Mean CV 8.7%
 
Inter-assay precision table
Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample. Samples were supernatants from PHA-treated PBMC cells.
Sample [CCL17]  (pg/mL) CV
1 2,773 8.7%
2 1,231 10.8%
3 647 13.3%
  Mean CV 11.0%
 
Spike and recovery

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample. Samples were supernatants from PHA-treated PBMC cells spiked with CCL17 recombinant protein.

Spiked % Recovery
IL6 (pg/mL) Diluent 5 RPMI+10% FBS
375 103% 99%
250 102% 106%
125 94% 85%


 

Dilutional linearity

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample. Samples were supernatants from PHA-treated PBMC cells. 

 

Sample dilution factor (x) Expected [CCL17] Observed [CCL17] Dilution Recovery (%)
  (pg/mL) (pg/mL)  
neat 4217.3 3840.4 91%
1/2 1692.3 1920.2 113%
1/4 813.0 960.1 118%
1/8 426.7 480.1 112%
1/16 236.8 240.0 101%
1/32 135.8 120.0 89%

 

 

Assay validation

Cell densities of PHA-stimulated PBMCs

Human PBMCs were stimulated with 10 µg/mL of PHA in RPMI for 24h, in a humidified atmosphere at 37°C and 5% CO2. Different cell densities were tested: 250, 500, and 750 kcells/cell were plated in a 96-well plate.

After 24h of stimulation, the cell supernatants were collected. 16 µL of each sample were transferred into a white detection plate (384 wells, low volume) to measure the secreted CCL17 in each sample. Then 4 µL of the HTRF human CCL17 detection reagents were added to the wells. After 2h, the signals were recorded for every sample.  

The CCL17 concentration in each supernatant sample was extrapolated from the standard curve prepared in RPMI.

As expected, the secretion of CCL17 increased with increasing PBMC cell density. 

assay validation PBMCs cell densities 62HCCL17PEG 62HCCL17PEH 62HCCL17PEJ.jpg

Dose response of PHA on PBMC

Human PBMCs (750,000 cells/well in RPMI) were stimulated with increasing concentrations of PHA for 24h, in a humidified atmosphere at 37°C and 5% CO2. After 24h of stimulation, the cell supernatants were collected.  16 µL of each sample were transferred into a white detection plate (384 wells, low volume) to measure the secreted CCL17 in each sample. Then 4 µL of the HTRF human CCL17 detection reagents were added to the wells. After 2h, the signals were recorded for every sample. The CCL17 concentration in each supernatant sample was extrapolated from the standard curve prepared in RPMI.

As expected, the CCL17 secretion increased with the PHA concentration, and reached a plateau (at around 30µg/mL).

 
Synergistic effect of co-stimulation of IL4 and LPS

Human PBMCs (750,000 cells/well in RPMI) were stimulated with 20 ng/mL of IL4 or 2 µg/mL of LPS, or co-stimulated with both IL4 and LPS. The plate was placed in a humidified atmosphere at 37°C and 5% CO2.  

After 24h of stimulation, the cell supernatants were collected.  

16 µL of each sample were transferred into a white detection plate (384 wells, low volume) to measure the secreted CCL17 in each sample. Then 4 µL of the HTRF human CCL17 detection reagents were added to the wells. After 2h, the signals were recorded for every sample. The CCL17 concentration in each supernatant sample was interpolated from the standard curve prepared in RPMI. 

IL4 stimulated the secretion of CCL17 in PBMCs, while LPS alone did not. However, there was a synergy effect from the co-stimulation of LPS and IL4, resulting in more secretion of CCL17 than with IL4 alone, in agreement with the literature (J. Immunol. (2017) 198 (9): 3426-3435). 

assay validation dose response 62HCCL17PEG 62HCCL17PEH 62HCCL17PEJ 1.jpg
assay validation dose response 62HCCL17PEG 62HCCL17PEH 62HCCL17PEJ 2.jpg

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