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Problem with ELISA false positives and noise - (Oct/07/2009 )

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Temperature should not influence CV unless the temperature was different across the area of the plate...or if you were comparing one assay to another done on different days. Your intra-assay CV is high.


sgt4boston on Oct 13 2009, 11:35 AM said:

I suspect it is not your equipment. I think you are pushing the limits of the assay. Please extend your curve into the ug and mg/ml area serial 1:3 or 1:10 dilutions and let us know if the %CV improves as the concentration increases.

Yes the %CV improves with increasing concentration. We have also had a recent run with lower %CV at low levels, (luck, practice, we're not sure). It will be a while before we get a chance to do the test in very controlled conditions, but I'll let you know how we go then.

We're still struggling with too high a background signal though. Anything from our protocol that could help reduce background signal?


Is there surfactant in your wash buffer? Tween -20 or Triton X 100 are usual additives.

You can allow the wash buffer to sit in wells for 10-15 seconds before you decand.

Be sure to vigorously decant and blot between washes.

After the last wash allow the plate to be inverted for a few minutes.

What is the %CV for each of the calibrator levels...including the new ug-mg/ml levels?

For example in a troponin test the CV is very high in the low pg ng/ml level and decreases to less than 10% as one gets to 5+ ng/ml levels.


So, our high CV% have mysterioulsy gone down to the point where triplicates now easily suffice. Which is nice for us but not so useful for the advancement of how to improve ELISA's as we don't know what is different now than before.

We're now going to try and reduce background by trying other blockers. So far we have tried standard stuff, skim milk, BSA, Ovalbumin. BSA has given the best responses so far. We're thinking of trying some of the more exotic non protein blockers or non mammalian blockers. Any recommendations either of what to try or how to try it?



For blocking try commercial products from Surmodics and East Coast Biologicals. There is also a blocking peptide fragment from Toyobo.

I suggest you also coat plates directly with ab. It may eliminate a potential source of problem.


Thank you, do you mean coat the plates directly with the antibody instead of using the streptavidin biotin system?

We tried that originally with maxisorb plates, and had varying results (maybe due to the unknown factor that has now disappeared). We could try that again now that the random noise seems to have gone and see whether it gives us a lower background noise.

I'll let you know how our blocking experiments go. My colleague has gone for a Pierce product Protein Free Blocker - I'll let you know how that goes too.

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