I have read somewhere that absorbance without stop solution is about half those with stop solution. Is this true?
Hello Marwa Ashraf Essa
No, I haven’t read something like that, ‘absorbance without stop solution is about half those with stop solution.’
I would like to mention that the addition of stop solution causes a drastic shift in pH and terminates the ongoing reaction. This stabilizes the color change of the medium, allowing a period in which the optical density of the assay can be accurately measured.
In ELISA, one must have OD readings in the linear range of the standard curve. For instance, standard of different concentrations is used for plotting a standard curve to measure the unknown samples. If stop solution is not used, then the signals from the required wells in ELISA will surpass the linear range of amplification and the OD readings will not be proper. So, you will end up with unexpected results.
Therefore, I would suggest you repeat the ELISA.
Best.
it depends upon which detection system is being used, but there are some where adding the stop buffer does both stabilize and increase the absorbance, whereas with others not. However I don't think you can rely on saying it is double.
You could find out how omitting the stop solution works in your system by repeating the experiment. In brief, set up your reactions in duplicate. Add the stop solution to one set and omit it from the other set. Get all the readings and compare the data. From there, you can determine the impact of omitting the solution and apply those findings to your current data set.
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