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AlphaLISA Human CCL17 Detection Kit, 5000 Assay Points

AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Human CCL17 Detection Kit, 5000 Assay Points
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image

The AlphaLISA™ Human CCL17 kit is designed for the simple and rapid quantification of soluble CCL17 (TARC)  in cell supernatants, providing a fast no-wash alternative to traditional wash-based ELISA assays.

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Feature Specification
Protocol Time 1.5h at RT
Sample Volume 5 µL
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The AlphaLISA™ Human CCL17 kit is designed for the simple and rapid quantification of soluble CCL17 (TARC)  in cell supernatants, providing a fast no-wash alternative to traditional wash-based ELISA assays.

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: 100 assay points
Part #:
AL3206HV
List Price
USD 1,035.00
Your online price:
Unit Size: 500 assay points
Part #:
AL3206C
List Price
USD 2,261.00
Your online price:
Unit Size: 5,000 assay points
Part #:
AL3206F
List Price
USD 14,900.00
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 14,900.00
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AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Human CCL17 Detection Kit, 5000 Assay Points
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead
AlphaLISA Human CCL17 Detection Kit, 5000 Assay Points
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead image
AlphaLISA Sandwich Anti-analyte Conjugated Acceptor Bead 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) seems to mediate the recruitment of regulatory T cells to sites of inflammation in patients with chronic hepatitis. Increased levels of serum CCL17 is associated with a greater risk of developing atherosclerosis.

Formats

  • Our 100 assay point kit allows you to run 100 wells in 96-well format, using a 100 µL reaction volume (10 µL of sample).
  • Our 500 assay point kit allows you to run 500 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample).
  • Our 5,000 assay point kit allows you to run 5,000 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample).

Features

  • No-wash steps, no separation steps
  • ELISA alternative technology
  • Sensitive detection
  • Broad sample compatibility
  • Small sample volume
  • Results in less than 3 hours

AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.

Specifications

Automation Compatible
Yes
Brand
AlphaLISA
Detection Modality
Alpha
Protocol Time
1.5h at RT
Sample Volume
5 µL
Shipping Conditions
Shipped in Blue Ice
Target
CCL17
Target Class
Cytokines
Target Species
Human
Technology
Alpha
Therapeutic Area
Inflammation
Unit Size
5,000 assay points

How it works

Principle of the AlphaLISA human CCL17 assay

The AlphaLISA human CCL17 assay is based on an AlphaLISA sandwich immunoassay involving a biotinylated anti-target antibody bound to Streptavidin-coated AlphaLISA Donor beads and an anti-target antibody conjugated to AlphaLISA Acceptor beads. Both antibodies are directed against the CCL17 protein. In the presence of the target, both antibodies bind to CCL17 and the beads come into proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer within the Acceptor beads, resulting in emission with λmax at 615 nm. The intensity of the signal is directly proportional to the concentration of CCL17 present in the sample.

Principle of the AlphaLISA human CCL17 assay
Protocol of the AlphaLISA human CCL17 assay
Protocol of the AlphaLISA human CCL17 assay


The AlphaLISA human CCL17 assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples (cell supernatants or fluids) or standards are dispensed directly into the assay plate for the detection of CCL17 by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Assay details

Human CCL17 assay details
Time to result 1.5 hours at RT
Kit component Lyophilized analyte, SA-Donor Beads, Biotinylated anti-analyte, Anti-analyte conjugated Acceptor Beads, Assay Buffer
Species Human only
LDL 0.3 pg/mL
LLOQ 0.9 pg/mL
Dynamic range 1-10,000 pg/mL
Calibration No international standard available

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 PBMCs.

Sample [CCL17] (pg/mL) CV
1 1389 10.1%
2 742 9.0%
3 305 6.7%
Mean CV 8.6%
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 PBMCs.

Sample [CCL17] (pg/mL) CV
1 1436 10.5%
2 696 14.6%
3 338 11.4%
Mean CV 12.1%
Spike and recovery 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 PBMCs spiked with CCL17 recombinant protein.

Spiked CCL17 (pg/mL) % Recovery
  DMEM+10% FBS RPMI+10% FBS 100% FBS
375 106% 99% 109%
250 101% 94% 111%
125 103% 91% 112%
Dilutional linearity 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 PBMCs.

Sample dilution factor (x) Expected CCL17] (pg/mL) Observed CCL17] (pg/mL) Dilution recovery (%)
neat 1431 1217 90%
1/2 562 608 109%
1/4 300 304 101%
1/8 148 152 102%
1/16 77 76 98%
1/32 37 38 103%
Selectivity table

Cross reactivities were assessed using recombinant CCL17 proteins from different species. Standard curves were generated for each protein diluted in the kit diluent for recombinant proteins.

Cross-reactivity percentage was computed by comparing the Alpha signal at the highest concentration (30,000 pg/mL) to the human protein. The assay is specific for human CCL17.

Proteins Cross reactivity (%)
mouse 0.00
rat 0.00

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/well were plated in a 96-well plate.

After 24h stimulation, the cell supernatants were collected and 5 µL of each sample was transferred into an AlphaPlate-384 to measure the secreted CCL17 in each sample. The standard curve was prepared in RPMI. All other assay components diluted in AlphaLISA Immunoassay Buffer were added into the wells following the kit protocol. After the final 30 minutes of incubation with Alpha Streptavidin Donor beads, the AlphaLISA counts were measured and the CCL17 concentration in each supernatant sample was interpolated from the standard curve.

As expected, the secretion of CCL17 increases with PBMCs cell density.

assay validation PBMC cell densities
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 atmospher3 at 37°C and 5% CO2. After 24h stimulation, the cell supernatants were collected and 5 µL of each sample was transferred into an AlphaPlate-384 to measure the secreted CCL17 in each sample. The standard curve was prepared in RPMI. All other assay components diluted in AlphaLISA Immunoassay Buffer were added into the wells using the kit protocol. After the final 30 minutes of incubation with Alpha Streptavidin Donor beads the AlphaLISA counts were measured and the CCL17 concentration in each supernatant sample was interpolated from the standard curve.  As expected, the CCL17 secretion increases with PHA concentrations and reaches a plateau (around 30µg/mL).

Dose response of PHA on PBMC
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 stimulation, the cell supernatants were collected and 5 µL of each sample was transferred into an AlphaPlate-384 to measure the secreted CCL17 in each sample. The standard curve was prepared in RPMI. All other assay components diluted in AlphaLISA Immunoassay Buffer were added into the wells using the kit protocol. After the final 30 minutes of incubation with Alpha Streptavidin Donor beads, the AlphaLISA counts were measured and the CCL17 concentration in each supernatant sample was extrapolated from the standard curve. IL4 stimulates the secretion of CCL17 in PBMCs while LPS alone does not. However, there is a synergy effect of the co-stimulation of LPS and IL4, resulting in more secretion of CCL17 than IL4 alone, in agreement with the literature (J. Immunol. (2017) 198 (9): 3426-3435 ).

assay validation LPS secretion

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