My students are culturing mixed mouse splenocytes in culture for 48 hours with anti-CD3/28. We are then going to analyse for the presence of Tregs and measure IL10 secretion by ELISA. In order to calculate what dilutions to use, I need to have some idea of what quantities of IL10 I should expect to detect. Does anyone know?
Depends on your ELISA and the standard curve end points. And it also depends on the mouse strain (inbred strains don't all respond the same) that you are using... and the cell concentratoin that you are plating per mL...
For most the highest concentration on the standard curve is 1000 or 2000 pg/mL.
Usually, you try to aim for the slope part of the curve on a 4-parameter graph.
The last time I did this, I only looked at IFNg.
If someone else doesn't have more updated information, the standard way to figure out dilution is to test it. For IL-10, I would plate the neat, 1/2 dilution and then 1/4. That should be enough.
Many thanks for your reply. These are C56Bl6, plated out at 10(6)/ml and the sensitivyt of the assay goes up to 2000pg/ml. I have plated out 1:2 and 1:10, so hopefully should be OK...
It's best validate and optimize your ELISA with titrated samples to determine the necessary concentraion of plasma, homogenate, media, etc. to plate. When we acquire new ELISA kits, we will optimize our kits by titrating our sample with 5 different concentrations and at a minimum of 3 technical replicates to validate a specific sample concentration for precision and reproducibility. Some of our injury models produce extreme amounts of our proteins of interest; it is important that our samples concentrations fall within the range of the kit’s standard curve. Here is a paper that may be helpful. It proposes a standard operating procedure for immunoassays and suggests some critical validation parameters. Hope this helps. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541289/
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