Immunofluorescent ELISA - (Oct/14/2003 )

Hi everyone,
Has anyone had any experience with ELISAs using immfluorescence. I'm using a biotynlated anti with an Alexa Fluor 488 strepavidin conjugate (10 ug/ml), problem is doesn't work even in biotin control wells. Any ideas welcome.
Yours desperately

Sounds like you're having fun! ELISA is a complicated art and I'm not sure I'll be a total help. First, I would check the binding of biotin to the plate using different amounts of biotin, different buffers, different pH, and different plates. Also, check different protein binding times and temperatures. These conditions can be worked out fairly quickly on a 96 well plate. Sometimes, high capacity/binding plates bind too much protein which can block antibody binding. A friend of mine had good luck with lower binding capacity plates. So you know, the protein can be rinsed off by vigorous washes as well.

Fluorescence may not be a good idea since many clear plates have crosstalk between wells and reflective properties. You'll have to use black opaque plates with optically clear bottoms. Alternatively, if you have a top reading device, you could use plates with black wells--I don't know if any of these are capable of binding peptide/protein. The read time of most devices also seems short. I don't know if the fluor can be adequately excited and measured in the short time.

Finally, it seems that your 488-SA is very high. I think 1 microgram/ml should be enough. You can test your machine for linearity/readout efficiency (and your plates for crosstalk) by diluting 488SA in PBS in replicates of 3.

One more thing....the fluorescent singal will be somewhat confined to a single plane on the plate. In many ELISAs that use enzymes, the whole 100-200 microliter solution has color/absorbance. I don't know if your detector can pick up that single plane (e.g. is it in focus).