What is the best method for measuring the levels of multiple cytokines and their changes over time in response to administering medicine?
Specifically, I am interested in monitoring changes in cytokine levels before and after administering a medication at 24h, 48h, 72h, and 96h time points. Which multiplex assay is most appropriate for this purpose (ELISA or RT-PCR cytokine panel), and what factors should be considered in choosing an assay?
Any advice or expertise on this topic would be greatly appreciated.
Regards..
ELISA would give you protein information, and RT-PCR would give transcriptional information. Changes in transcriptional levels does not necessarily indicate similar changes are occuring in protein, but can offer some information before pursuing protein work.
It is also possible to multiplex for cytokine production on the flow cytometer. There are readily available panels, and also customizable ones for both human and mouse.
ELISA could be helpful for longitudinal measurements as you could remove a sample of cell culture media at several timepoints. For PCR you would only get one time point. Note that for PCR you will also need a larger number of cells to get desired RNA concentrations, especially if assessing multiple gene transcripts.
It's depended on which part you would like to detect, cells or the culture sup.. For cells, you may need parallel samples for each time points. And it's easy to process qPCR to detect bunch of cytokines at the same time, Elisa for cell lysates is a little bit limited for this purpose, because you may need separate kits for each of cytokines. Like Sharmili said, you can also use flow to measure the cytokines, but you have to add reagent to stop the secretion. For supernatant, you can only use Elisa kits, but you don't need to prepare parallel samples for different points.
So I recommend using QPCR to detect cytokine mRNA level from cell RNAs after collecting all the time point samples.
Sharmili Vidyadaran
Lyudmila Akhmaltdinova
Sam E.J. Paton
Weiwei Shi
Thank you for all of your responses. In fact I intend to examine the cytokine level changes using isolated serum from the peripheral whole blood samples.
I would recommend Luminex technology as that would enable you measure several cytokines (multiplex) simultaneously. For example, one kit can enable you assay several cytokines at one shot. The only challenge would be with having the analyzer to help you read and assess the levels of each analyte following the bench work. ELISA would take a lot of time and you would need separate kits for each analyte. It may likely be more costly as well.
Hi Prabath
There is a kit in Biolegend, which has beads with intrinsic fluorescence used in various flow cytometers. It uses the principle of sandwich immunoassay. The advantage is you can quantify 13 human cytokines simultaneously.
https://www.biolegend.com/Files/Images/media_assets/pro_detail/datasheets/76100_Mouse_Inflammation_Panel_V05.pdf
Elispot would be a good technique to use for quantitative cytokine measurement
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