I did a few ELISAs 3 days ago (six 96 well plates) and some of them (about 10) were positive. My detector was an HRP conjugated antibody and I used TMB substrate. I stopped the reaction with sulphuric acid, and the wells turned yellow as they normally do. Today I had another look at the plates and they were completely clear, there was no hint of colour on them. Any idea why this might be? There is still liquid in the wells and no precipitate had formed, so it's not evaporation.
TMB is extremely light sensitive and developed plates should be kept in the dark. In fact, developed plates should be read within 1 hour at most as the colour fades with time.
Dear Gauri
Bear in mind that Elisa plates should be read immediately after adding H2SO4, as it will lose its optical density over time. Some Elisa protocols inform the optimal time frame when you should read your plate. The longer it takes, the more variable the color will be and your results wont be so reliable.
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