Cytokines are secreted by the effector cells of the immune system and play a major role in most chronic inflammatory diseases.
Quantification of cytokine is therefore a relevant biological target in the search for anti-inflammatory agents.
The determination of cytokines secreted by human mononuclear cells can be performed by ELISA or HTRF technology.
1. ELISA assay
The proteins to be assayed are bounded to a capture antibody then are detected by a biotin-streptavidin complex coupled to peroxidase, which in the presence of its substrate allows the development of a colorimetric reaction quantified by absorbance measurement at 450 nm.
The measured absorbance is proportional to the concentration of the cytokine measured.
2. HTRF assay (CISBIO)
Two fluorophores partners donor (europium cryptate) / acceptor (XL665) are linked respectively to an antibody specific to the protein to be assayed.
The time-resolved fluorescence energy transfer (TR-FRET) between the fluorophores is proportional to the concentration of the assayed cytokine present in the sample.
This assay is homogeneous and requires only small amounts of protein.
| TREATMENT | |||
|---|---|---|---|
| Format | 24 or 96 wells | ||
| Cellular model | Mononuclear cells isolated from human peripheral blood | ||
| Stimulation | LPS or PHA | ||
| Reference molecule | Dexamethasone | ||
| Incubation | 24H at 37°C under 5% CO2 | ||
| REVELATION | |||
| ELISA ASSAY | HTRF ASSAY | ||
| Format | 96 wells | 384 wells | |
| Detection | Absorbance measurement at 450 nm | Excitation wavelength : 337 nm Emission wavelength 1 : 615 nm (donor fluorophore emission) Emission wavelength 2: 665 nm (acceptor fluorophore emission) | |
Notes :
Recommendation :
Assays for TNF alpha, IL1 beta, IL-6 and IL-8 have been developed by ELISA.
TNF alpha, IL1 beta, IL-6, IL-8, IL-10 and INF gamma assays have been developed by HTRF.
Other cytokines can be tested on request.