Dear ResearchGate members,
As you know almost all of the commercially produced (available) kits that measure murine IL-6 by ELISA are designed basedon the Sandwich ELISA, that makes the kits very expensive. Has anyone done any other ELISA method to measure IL-6 (or any other similar interleukin or cytokine)? If so, could you provide some details on it, please?
Thanks for your help.
Hello Farbod Bahreini
The advantage of a sandwich ELISA is that it is more sensitive and robust as the antibody binds to two sites on the antigen. This increases the binding specificity of the primary capture antibody to the antigen as well as the binding specificity of the detection antibody to the antigen. The matched antibody pairs are tested specifically in sandwich ELISA to ensure that they detect different epitopes, for accurate results.
Many cytokines cannot be measured because they exist in serum at concentrations below the detection limits of conventional immunoassay methods, making it a challenge to fully characterize and apply physiological cytokine baselines. Although some cytokines are present at picomolar levels or above in body fluids, most of them are expressed at femtomolar levels or even lower. Many cytokines, which also includes IL-6, can be detected only during the acute phase when their levels are high.
Cytokine sandwich ELISAs are specific because antibodies are directed against two or more distinct epitopes. Therefore, sandwich ELISA is able to discriminate between cytokines that can have overlapping biological functions which I suppose are not resolvable by any other assay.
So, for reliable results, I always use sandwich ELISA for cytokines as its biggest advantage is its sensitivity (otherwise known as its detection limit). The sensitivity can increase dramatically if you have a high affinity and specificity of your primary as well as secondary antibody pair. Also, for the analysis of complex samples, sandwich ELISAs are particularly suitable, since the antigen need not be purified prior to measurement using this method.
Besides sandwich ELISA, PCR is another commonly used method for cytokine quantification which I would suggest.
Best.
Malcolm Nobre
Hi. Thank you so much for your response.
I agree that the sandwich ELISA is indeed more sensitive and more accurate. But suppose we measure the IL-6 derived from cell culturing. In that case, the sensibility is not a matter of issue as the cells we are interested in (J774 macrophage cell line) would secrete IL-6 upon activation. As we need to measure this frequently, the sandwich ELISA can be very expensive in the long term. Do you have any suggestions, please? For sure, qPCR is a good alternative, but we are primarily interested in the protein level and not the mRNA level.
I am sorry but am not familiar with mouse IL-6. I know that in human serum
its concentration is below 7 pg per ml. This can increase upon stimulation
by orders of magnitude. I would also prefer sandwich ELISA.