ELISA Software Background Cut-off or Threshold Calculation

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.

This is an example of an ELISA kit plate plate layout that can be used for calculating an accurate background cut-off value.

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.

ELISA Analysis Report Showing Background Cut-offs


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.

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