Hello,
I am using four parameter logistic (4-PL) curve fit (using GraphPad Prism8) to interpret my samples concentration after each ELISA assay. However, some of my samples with ODs less than the lowest standard still could be interpreted, which is totally illogic. So can anyone explain to me why does that happen?
I have attached some of my results as an example.
Thank you.
Dear Asmaa,
by fitting a curve you are not restricting to the area given by your samples. It runs from 0 to infinity mathematically speaking (probably works for negative numbers as well but unsure).
However, anything outside of the range of your measured standards should be read with care, you are not sure if the relation between OD and concentrations is different outside what you have measured.
Norman van Rhijn Thank you for your answer.
I thought that the samples could only be read by the software if they fall within the standards range. Especially that when I tried another type of curve fitting (Spline fit) it didn't recognize the OD values for samples with lower concentrations than the standards, which didn't happen when I used the 4-PL curve fit.
Correct! Spline is between the points and should connect them, the 4 parameter logistic regression ‘forms’ a curve given your data points. So difference is to be expected.
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