Using a fluorophore rather than a chromophore for ELISA detection seems like an obvious solution to increasing ELISA sensitivity, but I'm struggling to find work done in this area. Can anyone recommend a molecule sensitive to HRP/H2O2 that can give a bright fluorescence endpoint? We've tried fluorophore-labeled antibodies and saw no increase in sensitivity.
I have a flash-lamp sourced, monochromator-based fluorescence plate reader, not a laser-sourced one, so AlphaLISA is not a viable option.
Fluorogenic HRP substrates:
https://www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=133106&_bcs_=H4sIAAAAAAAAAMVSXU%2FCMBT9NX1xkWyrm%2BMRnIiR4OKi72W7sCbtStoOnL%2FeW8ZgiYTEF02a%2B9Wb%0Ac8%2B57W1A%2FCTTqmwKazwSxl4OescLMFfqlbVbQ%2BiEhDM8%2B%2F1%2BZCvQUq25QT8qlMRyY9BAjaZSEtAp%0AXeJlZaVACBJSd%2FzE6gZc7t%2F76AI%2FwM44HPvBDaYLtgLB640jwOSW0KmXgoXCclV7Pbsjoet86CTx%0Aew7oC2aZUJue4XuOhpVcIzRGwhI6K2RJaPr88bTE5pSbrWDtA7OwUbpFOlh8gRY7DmSHitZMmMuS%0AnI%2FvOmmP9VcrwcublbEaYf9dhiP2OxnoA0oDP3Z65kob0DjcVF4GWn3ykhnAh0vmbxkJx3%2Bl9Kwy%0At63Afxe7tItpmuNnErCAHYhLS%2BjkYOU1w2zNhXXD4y5wOEsmHcxALAmjcHoWfEidiY7CXTQ%2BVAcL%0A6CHdSLfH6LTZ6LTbaLjdH6%2FyDaBfQ5e4AwAA&OP=filter&filter=ft_6808/f_695306*&filterType=1&filterDispName=Fluorescent
Adam B Shapiro,
Thank you again for the help. I'm not sure what percentage of questions are answered by you, but if there were awards handed out for this kind of thing, you should be first in line.
QuantaBlu and Red were new to me, and searching for them led me to find this helpful paper (see Blais 2004) comparing several of these reagents. The most helpful images from that are attached here as well. I'm also attaching the product figure comparing Amplex UltraRed and AmplexRed.
Hi Oded, and thank you.
I have not, but see some listed. None are indicated for ELISA, which makes me wonder about their brightness in the assay. Have you used them successfully? Did they increase the sensitivity of your assay?
I used them in western blot they are much brighter then alexa fluor. Also look for company called biosynth they have product called aquaspark .it use hrp to produce fluorecent light
great, thank you. Somewhere in the files we have data from a fluorescently tagged secondary AB, and I'll see if I can find the relative brightnesses of these QD's and that fluorophore.
I'll check out biosynth as well, thanks
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