HTRF HUMAN IL2 KIT-Revvity

IL-2 plays a major role in modulating cell-mediated immunity through its effect on T cells. IL-2 promotes the differentiation of T cells into effector or memory T cells, thus helping the body fight off infections. IL-2 is also a key player in immuno-oncology where it serves as the main promoter of the differentiation of T cells into regulatory T cells (Tregs).

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. For more information on performance metrics or to learn more about AlphaLISA, check out the Human IL-2 AlphaLISA kit and the Human IL-2 AlphaLISA biotin-free kit. When assaying, always follow the recommended protocols and avoid highly hemolyzed samples.

Specifications

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 up to a 1536-well format by simply resizing each addition volume proportionally.

Principle of the HTRF Human IL2 assay

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.

Revvity also worked 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 IL-2 analysis.

Assay details

HTRF human IL-2 standard curve

The IL-2 standard curve is generated in the assay Diluent 5 provided in the kit.

HTRF Human IL2 standard curve

Human IL-2 assay specifications

Analytical performance

Intra and inter assay

Dilutional linearity

Spike and recovery

Cross reactivities

Cross reactivities were assessed using recombinant proteins from the IL-2 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 interpolate concentrations. The assay is human specific, as mouse IL-2 cytokines were not detected using this assay.

Assay validation

Effect of dexamethasone on PBMC induced IL-2 release

PBMC plated at 25, 50 and 100 kcells/well were stimulated for 18h with 50 ng/mL of PMA/ 1 µg/mL ionomycine and a co-treatment with dexamethasone (50 µg/mL). Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human IL-2 Assay kit.

Validation of HTRF Human IL2 kit on PBMCs Cell line co-treated with PMA/ionomycine and dexamethasone

Anti-CD3 and CD28 Ab dose response effect on IL-2 secretion

Jurkat cells plated at 200 kcells/well were stimulated for 24 h with increasing concentrations of anti CD3 and anti-CD28 antibodies : 0, 0.5, 1, 10 µg/mL for the anti CD3, and 0, 1, 5, 20 µg/mL for the anti CD28. 16 µL of supernatants were then transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP human IL-2 assay.

IL-2 secretion in PBMCs cells from different donors

PBMC plated at 100 kcells/well were stimulated for 18h with 50 ng/mL of PMA/ 1 µg/mL ionomycine. Then 16 µL of supernatants were transferred into a white detection plate (384 low volume) to be analyzed using the HTRF HP Human IL-2 Assay kit.

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In 2023, Revvity's turnover exceeded $2.7 billion. It has more than 11,000 employees worldwide and provides diverse services for customers such as pharmaceutical and biotechnology enterprises, diagnostic laboratories, scientific research institutions and government agencies. The company is a member of the S&P 500 Index, and its customers are spread across more than 190 countries and regions around the world.

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Cat.No	Name	Size
62HIL02PEH	HTRF HUMAN IL2 KIT	10K PTS

Specifications

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