My primary supervisor for my PhD is suggesting that if a sample is above the upper detection limit of a colorimetric ELISA, it is standard practice to dilute the sample wells after the stop solution reading and factoring this dilution into the concentration quantification. I've worked in immunoassays in industry for a year, never heard of this and know that practically TMB conversion will not be infinitely linear above the upper STD detection limit so this could mean a concentration of 15,000 and 100,000 above the upper detection limit could give the same absorbance, so diluting it would then also give the same absorbance but the results would indicate they're the same concentration. I really need reassurance that what she's suggesting is incredibly poor practise.
Hello Sophie,
It is not recommended. Instead, you optimize the sample dilution. For this purpose, you should experiment with different dilution ratios to find the optimal dilution for your samples. Minimize delay between adding the stop solution and reading the plate to avoid any further color development.
The purpose of dilution is to ensure that sample concentrations fall within the measurable range of the ELISA. This helps avoid signals that are too high (above the standard curve) or too low (below the detection limit), and it should be performed before the assay begins.
The stop solution is added to halt the enzymatic reaction and stabilize the signal, which is then read. Diluting the sample after adding the stop solution can alter the final absorbance value and introduce inaccuracies in your results.
As you mentioned, it is indeed a poor practice.
Best,
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