We are encountered with a phenomenon when T3 FREE was estimated in fish plasma by ELISA (DRG tests): some samples are shown no significant differences in total and diluted plasma (in PBS pH7.4, x10). Interesting that x5 dilution ratio of fish plasma in T3 and T4 study was fine. Does anybody encountered with such problem? How it could be cured? What buffer could be recommended for dilution fish plasma?
Are you seeing 'maximal' ELISA signal in the undiluted samples? It is common to find that undiluted plasma or serum can result in saturated ELISA signal -- sometimes it is 'true', if your analyte is present above the sensitive range of your assay, and sometimes it if 'false' where contents of the sample interfere with the assay (false positive or so-called 'matrix interference'). It sounds to me like you are having a matrix problem. There are so many variables that can interfere: different lipid content is a common natural problem, or lab/handling issues like hemolysis, trace clotting, insufficient/variable cell depletion, not to mention interference of the anticoagulant you are using.
Diluting into the linear range of the assay is the first requirement. If you think the 5x dilution is working, can you also go to 10x or 20x dilution and see similar 'appropriate' signal response? If so, such data suggests that you are getting an accurate answer.
When working with ELISA, it is always worth remembering that you have no guarantee of accurately detecting 'only' the molecule of interest, until you have truly proven that to yourself.
Craig,
Thank you for answer!
The samples of blood with we working to are have a high variability. We are not using anticoagulant, because of its interference. Some samples are close to sensitivity or to the linearity of test.
I’m also supposing that we have a difficult matrix that misrepresents the result. How it could be avoided?
How could I prove to myself that I'm working with the molecule of interest?
There were an experiment; we were diluted samples in few diluents in x5 and x10 times. The diluents were shown good linear range in x5 but not in x10, or conversely. We need an universally diluent, which could give “true” result in any dilution.
Hi Ekaterina,
Is this problem only happening when quantifying Free T3? If so, it could actually be an unfortunate side effect of the system itself. One of the Free T3 kits I have used in the past states: "Because of the nature of the T3 protein equilibrium, samples with high free T3 concentrations cannot be diluted with either serum or buffer and reassayed to obtain more precise free T3 values." I contacted the manufacturer and they confirmed that dilution will lead to erroneous results because of the binding chemistry.
I hope that helps!
Danielle
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