I'm trying to develop an immuno-PCR (ELISA, followed by PCR detection of oligo), but am struggling to find a compatible plate type. I've tried Nunc Top Yield strips from Thermo, which are supposed to be specifically made for immuno-PCR, but got no amplification when run on an ABI 7300. I think antibody coating would be poor in a regular PCR plate (made of polypropylene), and I want to avoid transferring samples (i.e. cleaved oligo) between an ELISA plate and a PCR plate to eliminate variability.
Does anyone have a suggestion of either a different plate type to try that will accommodate both ELISA and PCR, or a protocol for treating PCR plates to increase antibody binding?
I think that the plates are not the reason for your failed experiment. Nunc Top Yield strips seem to be specifically optimized for immuno-PCR. I would suggest to optimize the amplification conditions including several positive and negative controls for trouble shooting.
Michael G. Weller I've previously validated the oligo/primers using regular qPCR plates so I know that they should amplify just fine. However, I got no amplification in my PCR positive control (wells containing the oligo only, with no prior antibody treatment) when I tried the whole immuno-PCR protocol which tells me there's an issue with amplification. I think the main issue is that the Nunc Top Yield strips aren't making good contact with the heat block of the 7300 and so not getting to temperature, which is why I'm after a different plate type (specifically one that will fit well into a standard qPCR block).
Hi Taylor Pini , I was wondering if got your immuno-PCR work. If so, can you please share the details of what plate you end up using?
Seema Patel I didn't have any luck using my own system, so I tried a commercial product ("iqELISA" from Ray Biotech), but this didn't work at all either (not even the standards amplified in any recognisable pattern). Seems like a technology that's still in its infancy and needs a few more years to be developed, and some companies to invest some time and money to solve this issue!
Taylor Pini Thank you for the response, I am running into the problem of not having the right PCR machine that can fit the Nunc Top Yield Strip. We have a quant studio 5 qPCR machine but the Nunc Top Yield Strip won't fit in there. I was wondering if you ever tired doing the immuno-PCR with just the regular PCR tubes/strips?
Seema Patel I definitely found that same problem with the Nunc Top Yield Strips - I tried them in a 7300 and a Viia7 and never got any good amplification, I'm assuming because of poor contact with the heat block. I did try with regular PCR plates/tubes but the issue is that these use polypropylene which is hydrophobic and has very low protein binding capacity for antibody coating. I tried to remedy this by coating PCR plates with poly-l-lysine prior to coating with capture antibody, but never had any success with this. I think the solution is probably to run the immunocapture element in a plate suitable for ELISA, and then somehow cleave the bound oligo and transfer this to a PCR plate to run the PCR element. It would definitely introduce issues of potential oligo loss due to handling, but I don't really see another way around it.
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