Detection of communication molecules
Cytokines are vital signaling molecules, carrying messages between cells. They are released from one cell and subsequently bound by another cell. Since cytokines released from cells get diluted in the extracellular milieu, the detection method of this molecules has to be sensitive. In the case of inflammatory cytokines, their concentrations in the serum or plasma drops under 1 pg/ml. In contrast to traditional methods of proteomics, immunoassays are capable of detecting most of these low-abundance proteins. There are several methods for detecting and analyzing cytokines in a sample: traditional ELISA assays, enzyme-linked immunosorbent spot (ELIspot) assays, antibody array assays and bead-based assays.
Cytokines, being biomarkers for many disease, are essential players in the progression or regression of a pathological processes, thus cytokine assays are fundamental in disease diagnosing and monitoring.
In this short summary we are focused on the bead-based immunoassays, as they are most frequently used in our laboratory.
In bead-based multiplex immunoarrays, bead sets are stably coated with desired and specific capture antibodies. These beads are then incubated with a small sample volume, where the analyte particles bind specifically to the capture antibody. Following this, labeled detection antibodies bind to the capture antibody to form a sandwich consisting of detection antibody and analyte-capture antibody-bead complex. Passing such a complex through the detection system of flow cytometry allows for the identification and quantification of the desired compound. In comparison to ELISA, these cytokine multiplex assays in general are rapid, require smaller sample volumes and allow the simultaneous measurement of many different proteins.
Detremination of cytokines using available kits
Both the BD CBA (Cytometric Bead Array ) technology and LEGENDplex kits provided by BioLegend use fluorescent beads coated with capture antibody to quantify multiple proteins simultaneously in a single tube. The methodology significantly reduces sample volumes of analytes, when we compare it to the traditional ELISA or Western blot techniques. These kits can detect cytokines, chemokines and growth factors from serum, plasma and cell supernatant samples containing human, rat or mouse antigens. The principles of these assays can be found under the links below:
Detection of intracellular cytokines
- Stimulate cells e.g. PBMCs in vitro for 16h using CD3/CD28 antibodies, CMV peptide pool or SEB. Apply secretion inhibitors in parallel with stimulation factors.
- Perform intracellular staining. Recommendable solution is to use a buffer set and follow a protocol dedicated to cytokine staining, e.g. Cytofix/Cytoperm from BD.