4PL 1/y² fitting for Cisbio cytokine assays

The 4 Parameter Logistic (4PL) curve is the most commonly recommended curve for fitting an ELISA standard curve.

4PL 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. This is especially true in the low-end concentrations of the standard curve, where data points would be “lost” in a linear regression.

Don’t worry, you don’t need to have a degree in Statistics to use this equation and analyze your data. Software programs like Prism or Excel allow you to run a 4PL analysis without getting into the math, and there are free online software able to run this analysis for you.

 

4PL 1/y² fitting

Even though linear regression is easy to use and can be run with a very low number of standard points, it is not considered the best fitting method for biological phenomena like cytokine release, especially in an immunoassay.
The main drawback is that it is only applicable for samples that fall within the linear range of the assay, thus reducing analysis flexibility (dilutions …).

The 4PL equation includes 4 variable parameters related to the curve:

  • Estimated response at concentration zero
  • Estimated response at maximal signal
  • Slope factor
  • Mid-range concentration (or “point of inflexion”)

To get the most out of your data, we add a 1/y² weighting to the equation, thus making it a 4PL 1/y² fitting. The 1/y² correction basically considers the changes of variance occurring with an increase in signal.

With this fitting you get

  • A broader range of concentrations for analysis
  • Accuracy in the low/high ends of the standard curve

Please note that standard concentration is NOT in a logarithmic scale.

4PL 1/y2 in GraphPad Prism 7

If you are using GraphPad Prism 7: choose the "four parameters" equation available in the software.
Among the 2 equations suggested, we recommend the use of "four parameters" equation, the other "4PL" equation is NOT SUITED for analysing cytokine assays.

4PL 1/y2 in versions of GraphPad Prism prior to Version 7

The 4PL equation is already built into the software but if you have any doubts, you can download the following Prism file with the HTRF 4PL already built in.

Prism icon   Download HTRF 4PL Prism file

To enter the 1/y² weighting, follow the step-by-step procedure detailed in this video.

Free online software

To make your life easier, we have tested several free online software programs and we recommend MyAssays.com

This free online analysis software will give you access to easy data analysis, analysis customization and export capabilities.

HTRF data processing

HTRF technology uses donor and acceptor fluorophores.

Cisbio has developed a ratiometric measurement that uses both the emission wavelength of the donor (λ 620nm) and the acceptor (λ 665nm) to correct for well-to-well variability and signal quenching from assay components and media.

Emissions at 620 nm (donor) are used as an internal reference, while emissions at 665 nm (acceptor) are used as an indicator of the biological reaction being assessed.

For more information about data reduction, please visit ratio and data reduction

  • Ratio reflects the “raw signal”

The ratio must be calculated for each well individually. The 104-multiplication factor is introduced for easier data processing

  Channel A
665nm
(acceptor)
Channel B
620nm
(donor)
Ratio
(A/B) x 104
Std 0 861
939
1,199
17,393
18,529
18,652
668
669
696
Std 1 1,939
1,743
1,692
18,914
19,085
18,675
1,025
913
906
Std 2 2,859
2,941
2,822
18,108
19,509
18,034
1,579
1,508
1,565
  • Delta ratio reflects the specific signal

It is obtained by subtracting the background from the signal of each positive point.

  Channel A
665nm
(acceptor)
Channel B
620nm
(donor)
Ratio
(A/B) x 104
Delta ratio
Std 0 861
939
1,199
17,393
18,529
18,652
668
669
696
 
Std 1 1,939
1,743
1,692
18,914
19,085
18,675
1,025
913
906
271
Std 2 2,859
2,941
2,822
18,108
19,509
18,034
1,579
1,508
1,565
873

The Standard 0 (Negative control) plays the role of an internal assay control.

Delta Ratio = Standard or sample Ratio - Standard 0 Ratio

  • Mean and standard deviation can then be worked out from ratio replicates

  Channel A
665nm
(acceptor)
Channel B
620nm
(donor)
Ratio
(A/B) x 104
Delta ratio Mean ratio CV
Std 0 861
939
1,199
17,393
18,529
18,652
668
669
696
  678 2%
Std 1 1,939
1,743
1,692
18,914
19,085
18,675
1,025
913
906
271 948 8%
Std 2 2,859
2,941
2,822
18,108
19,509
18,034
1,579
1,508
1,565
873 1,550 2%