In the indirect ELISA being performed, when should the stop solution (H2SO4) be added after the addition of substrate (TMB)...?
It has been observed that adding H2SO4 after 2 minutes and 5 minutes resulted in standard curve with regression coefficient of 0.98, but with higher OD when H2SO4 was added after 5 minutes after the addition of TMB. Is there any way to standardize the procedure?
Most ELISA plate readers have an optical density range in which they are most sensitive (with our it is 1 - 1.5AU). If you do not incubate for long enough before adding stop solution, the low OD end of your curve will be less sensitive. Similarly, if you incubate too long then the high OD end of your curve will be less sensitive.
Find the most responsive OD range of your reader. If you have a significant concentration (a cutoff or normal range) then aim to get this in the middle of your OD range.
You can do this by running a few trials, adding stop solution at different times, or you can monitor the blue wavelength of the TMB on your whole plate and use that to decide when is the optimal time to add the stop solution.
I agree with richard. Beside you can see your negative control wells in your working plate on dark condition, when it starts changing color you can stop the reaction by adding H2SO4! Hope it would be of help.
You can test several timepoints: consider that the best incubation time will range from 5 to 15 minutes. Within this range you should obtain good results in any case; anyway, the best condition will be represented by the incubation time giving the higher "positive" vs blank OD ratio.
I can agree with Mohammad,
you have to add sulphuric acid just before the negative control begins to become colored
Color development reaction is a kinetic reaction in ELISA, so best time to stop reaction can be determined by measuring color density during different times after adding acid.Best time is the time when maximum color is achieved
The best time to stop the reaction is the moment when the negative controls start to become colored.
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