At the last step for data analysis, we always use a STOP solution to stop the TMB oxidized by HRP. Why do we have to stop the reaction? Couldn't we measure the color wavelength of chemiluminescence directly? Just like Western Blot data visualization, we just add substrates of HRP to see bands.
The reason for stopping the analytical assay is to make sure that you don't have overdevelopment which leads to precipitation and later would interfere with the OD readings. Overdeveloped wells will have a black deposition and will lead to extremely high or non specific absorbance readings which will show up as error instead of the actual values of OD.
Hello Cheng
We have to use stop solution in ELISA because ELISA technique is different from Western Blot. In ELISA, we have to have OD readings in a linear range. For instance, using standards of different concentrations for plotting a standard curve in order 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. As a consequence expected results will not be obtained. The function of the stop solution is to stop the on going reaction somewhere in the middle of the linear phase so that we get proper OD readings and thereby calculate the concentration of the unknown samples.
Thank you.
You do not need to stop the reaction if the color is not too strong, and if you have a fast reader, which avoids a time bias. This approach has some additional advantages:
1. You may repeat the measurement at different development times.
2. You may even repeat the last steps of the assay because the reagents are not denatured.
3. You can obtain a plot of the kinetics.
However, the yellow color shows about the double absorbance as the blue (unstopped) one.
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