I would like to quantify the activation of cell surface antigens, in this case CD69, on peripheral blood mononuclear cells (PBMCs) to screen for an immune response. Do I need to take any special steps or precautions, such as lysing the cells, since the antigens are found on the cell surface, instead of being secreted into the media?
As follow up, why is flow cytometry preferred over ELISA when quantifying CD cell surface proteins?
Why is flow preferred? Because the expression can be analyzed by a single cell basis. You can tell how many cells are positive and how highly expressed of this marker per cell. And the protocol is a LOT simpler..
I've don't know anyone who uses ELISAs for a cell surface marker. Due to the problem that you just listed... how do you get the markers off the membrane? It's not just lysing the cells. It should be a similar sample process as doing westerns on membrane bound proteins. There is a detergent step (I believe) to completely destroy the bilayer and ensure sufficent release of the membrane bound marker.
I'm pretty sure that the ELISA kits for CD69 should be intended for the soluable form, not the membrane bound form.
Dear it depends on the method adopted by the manufacturer of the ELISA Kits. Follow the instruction carefully and you will obtain a nice results.
Hi Natasha Worden , I agree with Shen-An Hwang, I don't know why you'd use ELISA to look for membrane-bound CD69. I would use Flow. I just wanted to add that you need to remember the kinetics of CD69 upregulation - it is among the first markers of T cell activation; it is upregulated in as little as 6 hours (depending on the stimulant), peaks around 24 hours and then down regulates & stays down. Other activation markers such as CD25 upregulate after activation & retain expression over time. However, CD25 does not peak until around 48 hours. So you need to take this into consideration when designing your experiment & measure the presence (or absence) of your marker at an appropriate time point. If you're looking for CD69 1 week after activation & concluding the cells are not activated, your conclusion would be incorrect. Hope this helps :) Melanie
Thanks for all the responses and good advice :). I do not have access to flow where I am, but I do have a plate reader, hence why I want to do ELISA. There are CD69 ELISA kits commercially available, so I supposed it was possible, but it may just be for the soluble form.
Natasha Worden I wonder if you could do an anti-CD69 ELISpot? The CD69 on the surface of the T cells MIGHT bind to the membrane-bound anti-CD69 & you may be able to quantify the frequency of cells that upregulate CD69? I don't know if that would answer the question you're asking. But ELISAs are definitely for secreted proteins :) Good luck
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA