I will be performing ELISA using plasma samples to determine certain lipids' concentrations. The protocol says 100uL of standard/sample must be added to 100uL of standard diluent, which is 1:2 dilution.
1)Should this dilution be considered during the preparation of initial dilutions?
2)What is the concentration of plasma to be diluted in the standard diluent during sample dilution before adding it to the well with 100uL standard diluent? If multiple dilutions must be checked for, what are the recommended dilutions?
Yes, you need to account for the 1:2 dilution factor when preparing your initial plasma sample dilutions.
The concentration of plasma to be diluted should be twice the concentration you want to achieve in the well after the 1:2 dilution.
For multiple dilutions, you can prepare a series of plasma dilutions (e.g., 1:2, 1:5, 1:10, 1:20) and then dilute each of these with the standard diluent in a 1:2 ratio before adding to the well.
By following these steps, you'll be able to determine the correct plasma concentration range and achieve reliable, reproducible results in your ELISA assay.
I’d first consult the literature to determine what your “expected” concentration range may be. Based on that information, I would choose a dilution factor in which the highest and lowest concentrations fall within the standard curve.
If you don’t have reliable information from the literature, you could run a partial plate, using 2 samples (1 expected high, 1 expected low). While the company suggests 1:2, Ive never really found this dilution optimal/helpful. I tend to run a standard curve, along with the following dilutions for each sample: 4X, 8X, 16X, 32X. After running the partial plate, it should become clear what the best dilution factor is.
Also, importantly, if the both the standards and samples are being added to 100uL of diluent when plated, then the relative dilution factor is still 1 (if the samples were added without prior dilution). If you wanted a 2X dilution, you would add 100uL of sample to 100ul of diluent. Then add 100uL of that solution to 100ul of diluent on the plate. But if the stnadards are not plated with diluent, this same dilution would be 4X.
I hope that makes sense. Please dont hesitate to reach out if you have questions.
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