I am running ELISAs. I run blank samples, without standard, and I subtract the mean OD value from the blank wells from the ODs of the other wells (standards and samples). Nevertheless, I have someone who insists that this is wrong. If I don't subtract the blank values, I don't understand the point of running blank samples at all? Your thoughts?
Hello Thierry Waltrich Augusto
I feel that the blank wells in ELISA will serve as a negative control. They are meant to analyze the contribution of background absorbance of the plastic and/or test reagents to the measured OD. It will allow the subtraction of background absorbance from the rest of the data points to ensure that one obtains the most accurate OD readings.
Therefore, some form of blank or background control should be run alongside standards and samples in every assay run.
Best.
It isn’t wrong but generally it isn’t necessary to subtract blanks. You can plot a standard curve with or without blank subtraction. Obviously you have to be consistent, and if your standard curve is blank subtracted, your sample has to be as well. One of the advantages of not subtracting blanks is that the signal to background ratio is clear to anyone looking at the standard curve, and that ratio is quite an important quality parameter.
In my experience a "Blank" well means a well receiving nothing but blocking buffer, sample diluent, and detector. A "Negative control" well receives the blocking buffer, negative sample, and detector. It is probably best to run both. I would not subtract things because you will want to see if there is any changes to your blanks and negative controls, rather then just subtracting them out of the data. Changes to the blank or negative control can show stability, or not, in the assay.
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