Hello, I am going to do an experiment about the estimation of the cellular immunity, specially that convey by T cells in immunosupressive viral infection. So, I am wondering Is there a simple method to estimate the cellular immunity like ELISA or others. Also, I want to know a good evaluation method for the whole immunity regardless cellular or humoral.
There is not a simple answer for this one. If I understand your experiment, you need to determine whether T cells are contributing to an immunosuppressive reaction to a viral infection. This would be unusual. The simplest method that can be easily done is to first observe the tissue for lymphocytic infiltrate with traditional histopathogy. If you see large infiltrates of lymphocytes, then you could perform additional staining via immunohistochemistry to identify which types of T cells are present (there are many subtypes and most have relatively unique surface markers, so they can be distinguished). If you have access to a flow cytometer, this is much faster and easier and you can stain for multiple subtypes of T cells all at once (CD4+, CD8+, Th17, etc.). ELISA and even ELISPOT for particular cytokines can give you clues as to whether your response is Th1/Th2 mediated, but probably will not give you what you are looking for.
Thanks for your reply but I need your help to explain this for me. If I use an immuno-potentiator and want to know its effect on the T cell response to viral infection which destroyed the B cell it is logic to measure the cytokines (IL6, IF ...etc) without histopathology or immunohistochemistery.
Christina. If I want to evaluate human immune responses, specifically allergic responses, would you suggest I do flow cytometry on peripheral blood leucocytes using the specific cell markers and the ELIZA to measure cytokines levels? Would this be adequate to capture changes in individuals with different environmental chemical exposures? Thanks.
Julia: it depends on the allergic responses, since you have different types of allergy responses (anaphylaxis and delayed-type hypersensitivities of various types). In general, a simple blood smear can allow you to count and differentiate leucocytes as lymphocytes, eosinophils, basophils, etc. and this can start to give you an idea of what type of allergic response you are dealing with.
For finer detail (CD4 versus CD8 T cell contributions) you would need to use flow cytometry, and peripheral blood is actually very easy to do, but this may not show localized immune responses: only systemic ones. In addition, your concentration of leucocytes should be highest in the affected tissue; once activated, leucocytes migrate through the bloodstream but then extravasate through the blood vessels and into the target tissue. What's circulating in the blood are essentially naive cells or cells-in-transit (that's my way of thinking about it). A skin-based allergic reaction would require a biopsy (and if anyone knows a great way to isolate cells from a skin biopsy, I'd love to know!).
ELISAs are good and reliable ways to measure cytokine levels, but there are also some good multi-analyte assays such as arrays from RayBiotech which allow you to do essentially simultaneously assay multiple target cytokines/chemokines/etc if you have the appropriate equipment.
Serageldeen:
You have a virus that infects and destroys B cells, and you want to know whether a particular compound affects T cell involvement in the immune response? I don't think that cytokine profiling alone is sufficient. T cells also physically interact and so cytokine responses would not include contact-mediated contributions. Furthermore, although certain types of T cells are known to produce certain cytokines (such as Th1/Th2 responses, which can be distinguished by production of IL-2/IFN-g and IL-4), trying to determine which cells are present, active, and contributing from just that information alone is tenuous at best. Noncanonical cytokine producers can mask and blur these responses as well (contributions from dendritic cells, etc.) I would suggest ELISPOT in lieu of ELISA in this case, since they will allow you to determine the number/frequency of cytokine producing cells for specific cytokines and you can purify your target cells first if need be.
Christina
I am grateful thanks your fruitful responses. I will do my best and hope that the results will be interesting
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