Hey there, I had similar problems a few months ago. As a first easy test, you should check that the HRP conjugated ab is cleaving the substrate. If that works, make sure your ELISA plate is binding an adequate amount of purified ab. That was my problem. We used evergreen plates, not costar and there was not sufficient purified ab binding. Hope this helps,

Alex

Hi Alex!

I have the same doubt about the HRP conjugated but do you know a simple test that I could do?

Also, I have some doubts about my substrate because after adding acetate buffer and H2O2 it turns withe and seems like some of the TMB starts to precipitate :S.

I´m using nunc maxisorp 96 well plates. Should I try costar?

Thank you!!!!

The way I test the conjugated ab is by adding 0.5-1ul directly to 1ml substrate and looking for color change. That should also tell you if your substrate is ok. We use a different system than yours I think, but I would try this simple test first to see if you have to troubleshoot any further. Also, I would check out the description of your plates online and see if they're coated to provide maximal ab binding. That was our problem, the evergreen plates were not meant for high efficiency binding, so we needed to use more purified ab.

-Alex

Nunc Maxisorp work well for ELISAs. I would try what Alexandra suggested. Also, I believe your H2O2 calculations are incorrect, by a factor of 10 (0.09% versus 0.009%). We use considerably less (2uL 30% H202 into 12.5mL=0.0048%). However, I'm not sure whether this would explain your results. What pH is your 0.1M acetate?

Hi Evan.

I think I´m having problem with my substrate solution (I hope is not the conjugated ab) because solution turns white and after a while you can see lumps or precipitation. My 0.1M acetate is on pH7.5...I guess this could be one of my problems. The protocol specifies pH 6.0 for acetate buffer. The substrate is being prepared as follows:

Dissolve 1.5mg TMB in 0.1ml DMSO. Then add 9.9ml of 0.1M acetate buffer (pH 6.0). Finally add hydrogen peroxide ti a concentration of 0.01% and filter through a whatman #1 paper filter. Solution is prepared fresh daily.

Your are right! my H2O2 calculations are incorrect, I used 30ul instead of 3ul. Thank you very much! I´ll correct it and I´ll adjust pH and try alex suggestion to see if it works.

I hope this is the problem. I´ll let you now all!

Thank you!!

1. Use a ready to use substrate: http://www.seramun.com/home.html

2. make sure that you have no cross reactions.

Your substrate should be colorless not yellow! Take stephan's advise and use a TMB substrate that contains stabilized peroxide.

Ramesh, I´m sorry, my bad! I write it wrong, here Is what really happened. Than you! :

Hello everyone!!!!

I corrected my formula as evan said and did the test that alex suggested and it worked great!!! Finally I got color. Thank you all!!

I just have one doubt left. I prepared my TMB working solution and after I added 1ul of anti Ig-Y (secondary conjugated antibody) directly to 1ml of this substrate solution...it turned orange. Then after adding sulfuric acid it turn yellow which is fine.

I was expecting it to turn blue. Does the orange means there was too much substrate for the enzyme so it tuned to orange and wasn´t enough to turn blue ?

Thank you.!

It is more likely due to too much enzyme. I've seen similar color changes when adding my Streptavidin:HRP solution directly to the TMB. It's nothing to worry about as your ELISAs will never have that much enzyme bound in any well to produce such a reaction.

Glad to hear everything has worked out for you