Good evening,
I have done several ELISA assays for my biomarkers, and I have observed that for some assays I get different concentrations from kits of different LOT numbers (but still from the same manufacturer). Meaning that there is a variation in the kit readings from batch to batch.
My samples are within the detection range of the kits, so I don't need to dilute them. However, I have found some opinions that samples need to be diluted anyway to reduce the matrix effect of the plasma/serum. So could that be a contributing factor to the varying results?
Thank you in advance.
Hi Asmaa,
If in the actual ELISA kit it states that it is siutable to measure your biomarker of interest in serum and or plasma then I would not dilute the samples. Especially if they are somewhere in the middle of your standard curve.
For some biomarkers you can buy WHO certified/reference standard with known amount of the biomarker in question. You can then run this standard together with your samples on every plate. It might help you identify if this is your sample or plate variability. Another advantage of using WHO certified standard is that you can report it in your paper which will be of great value for other researchers investigating the same biomarker.
If there are no WHO certified standards for what you are doing, you could pick one sample from your set and run it on every plate (it will act as your in-house standard).
Hope that helps,
Anna
ELISA from different lots and of different age may lead to different results (from the same samples). Thay may be dou to the age (esp. of buffers and controls), to specifity and activity of the antibodies used in ELISA (plate) etc.
Maybe you should work with relative values (compared to other measuremnts/times) instead of absolute valiúes with a quite high variability.
Anna Wawer and Andreas Eisenreich That was really helpful. Thank you so much for your answers :)
Yes ! Not for all but in some cases we need to dilute the samples before carrying out the assays. for example; for hsCRP we need to dilute the samples.
Ashok Kumar Shah yes I have realized that. So I am intending to compare the concentration values in diluted and undiluted samples for each biomarker to see whether there will be a difference.
Thank you for your contribution.
Chloe Guest These are biomarkers that are upregulated in plasma of heart failure patients.
Asmaa Elrakaybi So they are endogenous? How different are the concentrations you are getting between kits?
A parallelism experiment would be good to determine if there are any matrix effects that need to be diluted out.
This paper might be useful: Article Parallelism experiments to evaluate matrix effects, selectiv...
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