Cytokine Assays

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Disturbances in cytokine regulation are known to lead to inappropriate or ineffective immune responses. Cytokines are of particular interest in the fields of oncology, infectious disease, allergies and autoimmune diseases. Cisbio Bioassays developed a line of reagents for quantifying a variety of cytokines based on our patented HTRF® technology. These reagents produce quality results, are highly sensitive and have the flexibility to be used with cell supernatants or whole cells.

Features:
  • Compatible with cell supernatants and whole cells
  • Streamlined single-plate protocols for HTS
  • Monoclonal antibody-based for increased reproducibility
  • Highly sensitive (< 15 pg/mL)
  • Small sample size needed (down to 5µl)

Cytokines offer:
Applications:
  • Cytokine quantification
  • Infectious disease
  • Allergy studies
  • Autoimmune disorders

 

 


Assay Principle:

 

All cytokine assays can be run with cell supernatants or whole cells using the protocols shown here. Cytokines are measured using sandwich immunoassays involving two monoclonal antibodies: anti-cytokine MAb1 labeled with Eu-Cryptate and anti-cytokine MAb2 labeled with XL665. These antibodies may be pre-mixed and added as a single dispense step to further streamline the protocol. In addition, we have adapted our assay protocols to incubate the labeled antibody pair with cells during the stimulation step and eliminate the need to transfer cell supernatants prior to detection. The addition of potassium fluoride stops cell stimulation and allows the HTRF signal to be read immediately.



Product Specifications:

 

Cisbio Bioassays has developed a line of HTRF cytokine detection assays to address the screening of major inflammation targets. These specifications for these assays are summarized to the right.

  Human TNFα Human IFNγ Human IL1β Human-IL6 Human IL8*
Incubation 3 - 24 h at RT 18 - 20 h at RT 2 h at RT 3 h at RT 2 h at RT
Calibration range 0-2,000 pg/mL 0-1,500 pg/mL 0-2,000 pg/mL 0-2,500 pg/mL 0-2,000 pg/mL
Detection limit 10 pg/mL (0.6 pM) NIBSC 87/650 < 10 pg/mL NIBSC 88/606 < 15 pg/mL NIBSC 89/680 < 14 pg/mL NIBSC 86/680 15 pg/mL NIBSC 89/520
Extended range Up tp 6,000 pg/mL Up to 5,000 pg/mL Up to 10,000 pg/mL Up to 12,500 pg/mL Up to 5,000 pg/mL
* Available upon request

Assay Performance:

 

Cisbio Bioassays's assays are ideal for highly sensitive cytokine screening. They have rapid, easily miniaturized protocols that can be run in a single plate from cell stimulation through assay measurement. Figure 1 shows levels of TNFα (panel A), IFNγ (panel B), IL1β (panel C) and IL6 (panel D) detected using the cell-based and supernatant assay formats. These data show that both protocols give similar results and consistent cytokine concentrations with respect to the number of cells per well indicating that the presence of cells does not alter assay performance. Levels of IL6 (panel D) were not quantified above 6,250 cells per well as all concentrations were off the scale.

Figure 1: Cytokines were quantified using the appropriate HTRF® reagents. Increasing cell densities, ranging from 1562 to 100,000 cells per well, were distributed into a 96 well flat-bottom plate. To induce IL1β, IL6 and TNFα production, cells were stimulated with 10 µg/mL of LPS, while IFNγ secretion was stimulated by culture in the presence of 1ng/mL PMA and 500 ng/mL ionomycin. Cells were stimulated overnight at 37 °C. Cytokine detection was carried out following standard protocols. Standard curves were run in parallel. In cell based assays, 100 µl of potassium fluoride was added and the plate read. For supernatant assays, 50 µl of cell supernatant was transferred to the assay plate and incubated for the appropriate time with the corresponding antibody pair, before readout.

Ordering Info:

 

Cytokine Assay Reagents:

Description Quantity* Cat no. Product Insert
TNFα 1,000 tests 62TNFPEB pdf
TNFα bulk 20,000 test 62TNFPEC pdf
IFNγ 1,000 tests 62IFNPEB pdf
IFNγ bulk 20,000 tests 62IFNPEC pdf
IL1β 1,000 tests 62IL1PEB pdf
IL1β bulk 20,000 tests 62IL1PEC pdf
IL6 1,000 tests 62IL6PEB pdf
IL6 bulk 20,000 tests 62IL6PEC pdf
IL8 1,000 tests 62IL8PEB pdf
IL8 bulk 20,000 tests 62IL8PEC pdf
*based on 20 µl assay volume

Companion Products:

Description Cat no.
TNFα calibrator 62TNFCDA
TNFα control 62TNFTDA
IFNγ calibrator 62IFNCDA
IFNγ control 62IFNTDA
IL1β calibrator 62IL1CDA
IL1β control 62IL1TDA
IL6 calibrator 62IL6CDA
IL6 control 62IL6TDA


For more information or To Place an Order:
 

U.S. and Canada
1-888-963-4567

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Europe and other countries
+33(0)466 796 705

Japan
+81-3-5510-2932