If anyone has used ABTS peroxidase in ELISA as the substrate, how long do you incubate before you stop/read the plates? I am going to develop an ELISA and plan to use ABTS peroxidase and aim to read the plates after 15 minutes of incubation. Any thoughts/suggestions are highly welcome. Thank you.
Hello Tharaka Liyanage
The incubation has to be carried out for a much longer duration (30 mins) before you read the plate because ABTS is less sensitive than TMB or OPD in ELISA applications. It may take some time to properly develop its color. I would suggest you increase the incubation time to 30 minutes. Also, ABTS is not as often used as either OPD or TMB for ELISA. TMB is the most sensitive followed by OPD, and ABTS is the least sensitive. Another suggestion, why don't you change from ABTS to TMB?
Best.
Measure the plate every 5 minutes and determine the optimal incubation time (LOD, S/N, saturation, absolute value, accuracy (error), linearity if needed).
Malcolm Nobre Thank you very much for your thoughts. I have previous experience using ABTS peroxidase for dot blotting and Western blotting and thought to use the same in the new ELISA. Anyway there is no objection for me to switch into TMB :-D I will try both and see the difference. Thanks again.
Wolfgang Schechinger I was doing a similar approach these days measuring the plate every 5 minutes. Thank you very much for your thoughts.
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