hii there,
I would like to inform you that I'm working on IL-10 and IFN-gamma cytokines. I want to quantify these proteins levels in serum. I have tried using ELISA for the same but not getting any results. I have changed dilutions of primary antibodies from 1:1000 to 1:500 as well but still dint get colour. I have a limited quantity of serum so cant go for many changes in the protocol.
so if anyone else is also working on these cytokines and performing same technique, please enlighten me. If anyone could provide me protocol of indirect elisa, it would be great help for me.
Thanx.
Hello Vertica
Its clearly the best technique you need is Elisa, each kit have manufacturing guide to do..anyway try to avoid multiple thawing of the samples, check your procedure and the buffers that used, or simply use another type of ELISA kit because some companies or reservations conditions of the kit result in bad results...
Dear sir,
thank you for your reply. I'm not using kit as I have only 9 samples to validate my result, so I would like to know manual method of indirect elisa for quantification of cytokines in serum.
if you have any protocol, kindly provide me.
Indirect ELISA is used to measure antibody production in a qualitative or semi quantitative manner: plates are coated with an antigen and you test your sera for reactivity against that antigen (that is, presence of antibodies specific to the antigen).
For cytokine, non Ig proteins or other molecules (such as hormones), the choice is sandwich ELISA. This is a direct ELISA method in which plates are coated with a capture antibody for the molecule of interest and its results are quantitative (see attachment). Most kits are already standarized for the whole process and include a protein standard to validate quantitative results.
As above mentioned, the best choice is an ELISA kit for your cytokines at least you want to buy capture and detection antibodies for each cytokine to measure. There are options of "do it yourself" ELISA kits that include all the expensive reagents for the process and you only have to buy the plates and the work solutions (PBS-Tween, H2SO4, etc). Brands like Peprotech and eBiosciences sell these products.
Good luck.
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