When using ELISA, the assay range of the test is that of the dilutions made for the standard. Can we make more standard dilutions to have a wider assay range? or It will be wrong?
For most RUO ELISAs the dilutions recommended by the manufacturer are the highest and lowest ranges that can be used in that plate with an appropriate deviation. However, if you can go higher in analyte concentration without saturating the signal, or lower in analyte concentration without being too close to background then go ahead. Be careful, typically ELISA manufacturers have picked the dilution series accounting for inter and intra assay variation. So you will have to test multiple plates to make sure your new dilutions are reproducible. Also, be cautious that you are not altering clinically standardized ELISAs. For those types of assays the dilution ranges are set specifically to account for saturation points and deviation of the assay. So in those cases you should follow the protocol.
With commercial kits the assay range of the standard curve would be optiised for the conditions and generally should not be changed. However if you wish to increase the range why not increase the dilution of the samples?
Thanks Prof Walmsley, but my problem that most of my results had an OD lying below the lower detection limit. So, I want to expand the lower detection limit by more dilution of the standard, thus decreasing the gap between the blank (zero) and the lower test range. Is it OK or will be wrong?
I may not understand the issue correctly, but it seems you could use your current standard curve equation to solve for the unknown sample concentration, even if the sample has a lower OD than the most dilute sample. Increasing assay sensitivity, or perhaps attempting to lower background, may also make it easier to quantify low concentration samples.
If it a non clinical assay you can as low as you wish. Just keep going lower in dilution, keeping in mind the above comments. Again the only issue is your deviation, if you have issues with your std deviation, try running multiple dilution curve standards and multiple analyze samples on the same plate. This will help getting accurate results at lower concentrations.
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