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.

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

Intra and inter assay

Dilutional linearity

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.

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.

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.