IBMX Phosphodiesterase inhibitor
The HTRF human IFN gamma kit is designed for the quantification of human IFN gamma release in cell supernatant.
IFN gamma is a pro-inflammatory cytokine secreted by T and NK cells in response to viral infection and other inflammatory conditions. IFN gamma plays a major role in innate and adaptive immunity by promoting T cell differentiation into TH1 and the development of Treg, as well as by inhibiting T cell differentiation into TH17. Its anti-tumor properties have been used in the treatment of osteoporosis and it is of great interest in immuno-oncology where it improves survival in patients with bladder or ovarian carcinoma.
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 IFN-γ AlphaLISA kitand the Human IFN-γ biotin-free 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.
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 Cisbio 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.
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 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 IFN gamma analysis.
|Sample size||16 µL|
|Final assay volume||20 µL|
|Kit components||Lyophilized standard, frozen detection antibodies, buffers &protocol.|
|LOD &LOQ (in Diluent)||14 pg/mL &21 pg/mL|
|Range||21 – 4,000 pg/mL|
|Time to result||Overnight at RT|
|Calibration||NIBSC (82/587) value (IU/mL) = 0,019 x HTRF hIFN? value (pg/mL)|
|Sample||Mean [IFN?] (pg/mL)||CV|
Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.
|Sample||[IFNg] (pg/mL)||Mean (delta R)||CV|
Each of the samples was measured in 4 different experiments, and % CV was calculated for each sample.
The excellent % of recovery obtained from these experiments show the good linearity of the assay.
|Dilution factor||[IFNg] expected (pg/mL)||[IFNg] detected (pg/mL)||Recovery|
|Sample||[IFNg] added (pg/mL)||[IFNg] expected (pg/mL)||[IFNg] detected (pg/mL)||Recovery|
The same amount of recombinant cytokine was added to 2 different serum samples, and 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.
PBMC plated at 50, 100, 200 and 400 kcells/well were stimulated for 3h with increasing concentrations of PMA (0, 1, 50 ng/mL) added to a 500 ng/mL Ionomycin solution. 16 µL of supernatants were then transferred into a white detection plate (384, low volume) to be analyzed with the Human IFN? Assay.
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