The ELISA Analysis 4PL and 5PL curve fitting models are based on the “Analysis of dose-response curves” (Package ‘drc’) statistical programming package for biology and other fields that leverage dose-response curves. This package is part of the open source R statistical software environment. Further details on the model can be found in the following references:
- The following is a specific reference for the Package drc:
Ritz, C. & Streibig, J. C. (2005) Bioassay Analysis using R. J. Statist. Software, Vol 12, Issue 5.
- The following papers are cited by the Package drc creators:
4PL curve: Seber, G. A. F. and Wild, C. J (1989) Nonlinear Regression, New York: Wiley \& Sons (p. 330).”
5PL curve: Finney, D. J. (1979) Bioassay and the Practise of Statistical Inference, Int. Statist. Rev. , 47, 1-12.”
ELISA kits offer a reliable and repeatable method for detecting any given target in may types of sample. However ELISA kits involve several steps and reagents and in some cases significant background noise can result from the interaction of these reagents. For this reason it is important to ensure that your results are statistically significant when compared to the natural background noise of the ELISA system.
The ELISA Analysis platform calculates the background cut-off automatically whenever you include negative controls in your experiment. Here is how it works:
1. Specify the wells that are negative controls in your plate layout. The more negatives you include the better as this will increase the statistical accuracy of the cut-off calculation.
2. When you click “Analyze & Create Report” we will take the negative OD values, calculate the mean OD for the negatives and calculate the standard deviation (SD) for the negatives. The background cut-off is then calculated as the mean plus three times the standard deviation:
Background Cut-off = Mean of Negatives + (3 x SD of Negatives)
3. In your ELISA Analysis report you will see that the Cut-off Threshold is displayed at the top along with the Baseline value which is equal to the mean of the negative values. Where a mean OD reading is below the Background Cut-off value then the calculated Mean Concentration is not provided and there is a cross marked in the “Excluded: Optical Density below cutoff” column.
Note: If you do not include any negative wells in your plate layout then the Background cut-off value will default to zero.
We hope that you will find this article useful. Please feel free to contact us with any questions and feedback regarding this feature and how we could improve it.