I am optimizing detection of an antimicrobial peptide using a sandwich ELISA. The standard is diluted in 2% BSA buffer and the standard curve is satisfactory. However, now that I am trying to optimize the use of undiluted biological fluids such as skimmed breast milk and plasma in this assay, I have difficulty measuring the peptide. In case there is simply no peptide to be detected in the samples, I have tried 'spiking' the fluids with the standard and detected a much lower than expected concentration using the assay. I have also found that diluting the fluids can improve recovery, though this is not ideal since I expect low concentrations to be measured. Can anyone suggest a way to improve detection? I am currently incubating the samples for 1 hour at room temperature.
Did you try to centrifuge your samples and use the supernatant for the assay?It looks like there is something in your samples that is interfering with the assay. Usually we use dilutions or heating. In your case you are studying proteins so heating is not an option. Also check the PH of your samples and compare it to the kit optimal PH.
Thanks Sulaiman. You are correct that heating is not an option. We did not use a kit (we developed the assay ourselves) so I am unsure of the optimal pH for the assay. We centrifuged the samples to remove the fat from milk and cells from plasma.
Dear Stephanie,
Your home made assay is perhaps showing a "Hook effect". In this case, the high concentration of peptide is underestimated. To verify this hypothesis, you can dilute your sample many times. The measurement of each dilution will give you an apparent progressive elevation with the first step of dilution and finally in diluted samples, you will retrieve a progressive decrease of the peptide concentration. In this final area of dilution you can then work classically.
Can you increase the sensitivity of the assay by using a different detection method e.g. chemiluminescence/fluorescence? Then samples can be diluted (reduce interference) but still detected at the lower concentrations?
Not sure of current detection method or expected protein concentrations so this suggestion might be totally irrelevant....
Maybe try preabsorption of samples?
Hi Stephanie,
you need in most cases your dilution/sample buffer. This buffer has some functions: The detergent (Tween 20 or 40 at 0.1% v/v) reduces unspecific hydrophobic interactions either with the solid phase or the solid phase bound proteins/antibodies. The pH is constant, sometimes some protease inhibitors are helpful, in many cases I increase the salt concentration from physiological 0.15 mol/L up to 0.3 mol/L. This will suppress low affinity unspecific reactions ...
The hook effect was already mentioned. To make sure that you didn't have this problem, run serial dilutions of your sample. You need to find an optimal dilution anyhow.
In general, I suggest to check some protease / peptidase inhibitors. Those enzymes are mostly very active in biological samples.
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