Hello! Has anybody used Biotin Tyramide (B-T) for signal amplification in an ELISA procedure? What was the optional concentration (at least a range)? Was it a ready made kit or home-made B-T?
Thanks a lot!
I have used biotin amplification in ELISA development. Based on my experience, I would not recommend doing this, but then I don't have a whole lot of information about your specific situation & application.
I was developing an assay on the xMAP platform, and was producing MFI's around 3,500 for 625 pg/mL standards. A benchmark assay was producing closer to 18,000 at that analyte concentration. Biotin amplification was investigated as a method to close this gap, and in that sense, it was successful. Biotin amplified standard curves reached over 20,000 MFI.
However, this was not an a real increase in assay sensitivity, or resolution. In fact, this assay had substantially lower resolution than the benchmark assay, even though the same MFI range was covered. The reason for this is that there was no increase in measurement points, i.e. antibody binding sites.
By analogy, if you put a magnifying glass over $1, you don't get $10.
Could you give any information regarding why you want to pursue biotin amplification? If you are worried about assay signal strength, I advise you to look to your antibodies first. They are fundamentally the most important component of your ELISA, and all quality (or lack thereof) in the final assay will depend on the quality of your antibodies.
Dear Karl, thank you for your reply. This is my original topic on ELISA for more info: https://www.researchgate.net/post/How_to_increase_the_ELISA_sensitivity
So, as you can my Abs work but I really need higher sensitivity/stronger signal. All the techniques and tips I tried didn't work (I have also used amplification solutions from a company, they didn't work either, sensitivity remained the same). So, BT seems to be my last resort, since I already have these Abs (and not to purchase a kit).
in my opinion the antibody is the key factor to your assay. if you used biotin to amplificate your sensitivity, the molar ratio (biotin per antibody) is important! my suggesttion is 40-80 fold. i hope it can help you.
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