Hi everybody,
I need to develop an ELISA and I just wonder whether anyone can shed some light on the sensitivity of a fluorescent ELISA (detection with a fluorescent dye-labelled detection antibody), compared to the regular ELISA (HRP-labelled detection antibody).
With our plate reader both detection method is feasible, and I think that the sensitivity could be similar (of course it depends on the plate reader, the dye itself and it's amount on the detection antibody etc...) while the handling is easier with the fluorescent version (no additional incubation is needed at the end, also doesn't need to stop the reaction).
I'd appreciate if you share your thoughts and experience with me.
Thank you,
BR,
Daniel
Enzymatic detection is more sensitive than fluorescence detection because of the amplification resulting from multiple catalytic turnovers of the enzyme. If you use a fluorogenic or especially a chemiluminescent substrate, the sensitivity will be greater than for a chromogenic substrate. Here is a guide:
https://assets.thermofisher.com/TFS-Assets%2FLSG%2FApplication-Notes%2FTR0033-Substrates-ELISA.pdf
Hi Adam, thank you very much for your answer!
I also thought that the amplification gives a lot to it, but some plate reader has a super sensitivity nowadays for fluorescence.
Based on your recommendation I think I'll make a comparison of various (fluorescent, chemiluminescent, colorimetric) ELISA substrates. Thank you for the list!
BR,
Daniel
Well, you don't always need the highest sensitivity, it depends on the usual concentration range of your analyte. Colorimetric assays are popular because they are cheap, and this is important for commercial assays, but for in-house testing maybe fluorescence makes handling easier. And, as mentioned, luminescence gives you the best sensitivity available, so if you work at very low analyte concentrations, luminescence is the way to go.