What does ED50 Mean?
What does ED50 mean?
We all think we know what the ED50 value of a recombinant protein is; in short, it is the concentration at which the protein exhibits 50% of its maximum activity. Simple enough, right? It is our go-to piece of information on the data sheet when we consider a protein for purchase, but how many of us are truly familiar with what it means? Below are some guidelines for understanding the uses and limitations of ED50 values.
ED50 is assay-specific
The cells you use in your bioassay and the readout that you are looking for both influence the ED50 of the protein you are testing. Many proteins have diverse biological functions and can be used to elicit a wide variety of responses in different cell types. A protein that is a potent mitogen for one cell type may be a weaker inducer of cytokine secretion in another. These assays may be both biologically relevant, but the ED50 values that they generate cannot be directly compared to one another.
ED50 can vary between experiments
Once you are working with the same cell line and looking for the same readout, you should expect to get identical results each time, right? We wish it were that simple, but the reality is that small changes in protocol, cell culture conditions or passage number, etc, can make a big difference in your results. It is not at all unusual to see a 2- to 10-fold difference in ED50 between experiments, even when using the same lot of protein and the same cell line. For this reason ED50 values are best reported as a range rather than a fixed number.
What about specific activity Units?
No need to panic when the publication you are referencing reports recombinant amounts in Units, rather than concentration! A simple calculation will convert your ED50 to Units/mg. Keep in mind that the assay- and experiment-dependency of these values still apply.
1x10e6 / ED50 in ng/mL = Units/mg
Is ED50 always the best measure of activity?
ED50 is most applicable to proteins with sigmoid activity curves where the 50% concentration value can be easily identified, as in the example below. Bioassays with bell-shaped activity curves or where the read-out is qualitative, rather than quantitative, are best described in terms of maximum activity, or range of activity. Keep in mind that not all proteins will have activity curves that plateau with increasing concentrations. It is often the case that proliferation assays for many growth factors will show a decrease after maximum proliferation has been reached. The activity curve is still a sigmoid curve and ED50 can still be applicable, just know that more of a protein is not necessarily a good thing when designing your experiments.
Proliferation of M-NFS-60 cells in response to Mouse M-CSF Recombinant Protein, as measured by MTT (proliferation) assay. The ED50 is clearly visible for proteins with sigmoid curves such as this one.
So why is ED50 useful?
After all of these limitations, you may be left wondering how ED50 is useful at all! However, the ED50 value lets you know that the protein has been tested in a manner that is biologically relevant. It also gives you a starting place for your experiments- but remember you will need to find your optimal concentrations (and even ED50) for your assay.
Lastly, the ED50 establishes quantitative criteria for the side-by-side comparison of two proteins. This allows eBioscience to ensure that each new lot performs as well as the next, so you can feel confident that you can have fewer variables in your experiments. This information is available to you; just ask our Tech Support Experts!