I am planning to run an ELISA soon but I have a question regarding sample preparation. If I don’t know the working range of my samples for the ELISA, should I just add the samples to the plate instead of diluting them and use the standard curve to measure against this? Or should I initially run the assay with a series of dilutions to calculate the working range, although time and expenses are a limiting factor with this method?
The ideal way would be to test several dilutions of some samples at the beginning to know which are useful. If your samples are too concentrated, saturation will preclude a proper estimation of the concentrations and all of them will probably look the same. If they are too diluted, the sensitivity could be not enough to detect the analyte.
I would always dilute the samples in the same diluent used for the standard (assuming that it contains blocking agent(s)), at least 1/2, to avoid non-secific background. But is it totally impossible for you to have a gross idea of the expected concentrations in the samples? Probably you can at least predict that and select the dilution range on these bases.
I agree with Gertrudis Rojas, the ideal way would be to test several dilutions (for example 1/2, 1/4 and 1/8) of some samples at the beginning to know which are useful.
I agree with Gertrudis_Rojas. In my case I would go for criss-cross dilution where I take several dilutions of antigen in column and several dilutions of primary antibody.
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