I have been trying to detect cytokines (IL-2, IL-10, IL-6, TNFa, IFNg, IL-17A, IL-4) in human serum from healthy individuals plus a Parkinson's disease patient using CBA, but I cannot detect anything (even in undiluted serum). I have been told by BD that the cytokine levels in serum from healthy individuals is too low to detect by CBA. However, cytokine levels are increased in PD, so I would expect to detect some.
Is there any way to concentrate serum samples? Or to increase the sensitivity of the CBA assay?
Are you limited to CBA? high-sensitivity luminex kits and the like (we use Milliplex) should give you enough sensitivity for at least some of those. It will be hard to derive quantitative analysis values from comparing negative measurements to positive.
I have done CBA on splenocytes,,,cryo preserved pbmcs,.stimulating with super antigen,.i managed to detect the cytokines you mentioned, .with both Elispot and cba
Please consider that cytokines are small molecules and the biochemical or physical filters (such as the lung and the kidney) dramatically reduce the concentration of circulating cytokines. Thus, in vivo measurement of circulating cytokines or chemokines in patients' sera suffer this severe bias. On the contrary, the measurement of these molecules in cell culture is extremely accurate due to the absence of any kind of filter, being only the difference between the molecules secreted and the molecules removed by the presence of (specific) receptors. Tests that are excellent in vitro may have thus problems (not due to the test, but to the biology of the system), in sera or plasmas. For all these reasons, often, an increased concentration of cytokines in serum is just a expensive proof of a reduced function of kidney !!!!
I use Milliplex and ELISA assays and I have not found problems in measurements.
I have used CBA for human semen cytokines, TNFa, IL1b, IL6 and IL8. Worked fine on most samples.
However, since the immune system and response change their properties with development, differences in patients' age should be taken into account, both in physiological and in pathological conditions. I just the properties for your samples. the was a study by Giulio Kleiner tittled: "Cytokine Levels in the Serum of Healthy Subjects" they show that cytokine detection in human samples may be influenced by several factors such as age.
How much are you diluting your serum samples? Using fewer beads than suggested can boost your sensitivity (i.e. BD may say to use 5000 beads/analyte/well. Use 1500 instead).
In fact Evan Chiswick , with less beads (± 2500 beads) increased the sensibility in mean 15% with human adipose tissue and plasm samples. Excellent recomendation. This way you can optimize the numbers of samples per kit.
Evan Chiswick Rafael Ferraz-Bannitz great suggestion!
We used half of the recommended CBA beads and measured cytokines in serum samples using FACS Calibur. We noticed slightly higher sensitivity and really improved specificity, especially with beads located close to each other in alphanumeric position designation (A7, B7 and B8).