Stop Solution Doesn't Stop the Reaction - (Sep/28/2005 )
Hello Everyone:
I am currently running an indirect ELISA. My steps included: Coat w/ Ag, 3x wash, block with blocking buffer, incubate 1 hr, 3x wash, add test Abs, incubate 1 hr, 3x wash, add 2nd Ab labeled with HRP, incubate 1 hr, 3x wash, add ABTS (liquid form), incubate 15 minutes, stop with 3N H2SO4, read plate. The problem I am experiencing is that when I add the stop solution there appears to be some color reaction taking place. I often find that within 5 to 15 minutes after adding the stop solution the whole plate appearance changes...some wells are darker than others. This happens even among triplicate test samples and the controls often have color. Prior to adding the stop solution everything looks fine. Is my stop solution not strong enough? Do I really need to use a stop solution? Any advice is greatly appreaciated 
Is it a 'home-made' protocol or from a company. Sometimes it helps when you shake the plate after adding the stop solution. Normally we use 2 N H2SO4 and that works fine for us.
A question from my hand.. does the ABTS had anything to do with it? Why are you adding this and could you try the ELISA without it?
Hi
I don't know much about ABTS BUT I do know you need to incubate it in the dark. Also its an acid so I dont think sulphuric acid is going to stop the reaction. Leave for 15min then read plate without stop solution. Alternatively do as most people do and use TMB. Its a good substrate (usually colourless to blue then to yellow after stop) and you get a good increase in signal when you add the sulphuric acid. Read it at 450nm. Oh and its not light sensitive.
tara
Parky
Nop stop
I am currently running an indirect ELISA. My steps included: Coat w/ Ag, 3x wash, block with blocking buffer, incubate 1 hr, 3x wash, add test Abs, incubate 1 hr, 3x wash, add 2nd Ab labeled with HRP, incubate 1 hr, 3x wash, add ABTS (liquid form), incubate 15 minutes, stop with 3N H2SO4, read plate. The problem I am experiencing is that when I add the stop solution there appears to be some color reaction taking place. I often find that within 5 to 15 minutes after adding the stop solution the whole plate appearance changes...some wells are darker than others. This happens even among triplicate test samples and the controls often have color. Prior to adding the stop solution everything looks fine. Is my stop solution not strong enough? Do I really need to use a stop solution? Any advice is greatly appreaciated
Thanks Parky....didn't realize that I was trying to stop an acid with an acid...Duh. I called the manufacturer and they suggested 2 to 3 N NaOH. I will try that today. Thanks again.
I don't know much about ABTS BUT I do know you need to incubate it in the dark. Also its an acid so I dont think sulphuric acid is going to stop the reaction. Leave for 15min then read plate without stop solution. Alternatively do as most people do and use TMB. Its a good substrate (usually colourless to blue then to yellow after stop) and you get a good increase in signal when you add the sulphuric acid. Read it at 450nm. Oh and its not light sensitive.
tara
Parky
Nop stop
Hello Everyone:
I am currently running an indirect ELISA. My steps included: Coat w/ Ag, 3x wash, block with blocking buffer, incubate 1 hr, 3x wash, add test Abs, incubate 1 hr, 3x wash, add 2nd Ab labeled with HRP, incubate 1 hr, 3x wash, add ABTS (liquid form), incubate 15 minutes, stop with 3N H2SO4, read plate. The problem I am experiencing is that when I add the stop solution there appears to be some color reaction taking place. I often find that within 5 to 15 minutes after adding the stop solution the whole plate appearance changes...some wells are darker than others. This happens even among triplicate test samples and the controls often have color. Prior to adding the stop solution everything looks fine. Is my stop solution not strong enough? Do I really need to use a stop solution? Any advice is greatly appreaciated