Principle of the HTRF HP human IL-6 assay

Cell supernatant sample, or standard is dispensed directly into the assay plate for the detection by HTRFTM 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.

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.

HTRF HP human IL-6 standard curve 

The HP human IL-6 standard curve is generated in the assay Diluent 5 provided in the kit

Sample size	16 µL
Final assay volume	20 µL
Kit components	Lyophilized standard, frozen detection antibodies, buffers & protocol
LOD & LOQ (in Diluent)	10 pg/mL & 16 pg/mL
LOD & LOQ (in DMEM+10%FCS)	16 & 24 pg/mL
LOD & LOQ (in RPMI+10%FCS)	11 & 21 pg/mL
Range	38.8 - 12000 pg/mL
Time to result	2h at RT
Correspondance to I.S.	NIBSC (89/548) value (IU/mL) = 0,1 x HTRF HP hIL6 value (pg/mL)
Species	Human only

Intra-assay precision table

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Inter-assay precision table

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample.

Dilutional linearity

The recovery percentages obtained from these experiments show the good linearity of the assay. 

Spike and 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.

Cross reactivities

Cross reactivities were assessed using recombinant proteins from the IL-6 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.