Cytokines are a broad class of soluble proteins, glycoproteins, and peptides that act as chemical messengers of the immune system. These small proteins are essential intercellular communicators that carry messages from one cell to another and contribute to cell growth and immune response.
Cytokines include chemokines, interferons, interleukins, lymphokines and cannot cross the lipid bilayer of the cell wall to enter the cytoplasm. They have been shown to be key players in endocrine, autocrine and paracrine signaling as immunomodulating agents.
Since cytokines help to regulate important functions in the human body, researchers and clinicians have put forth a great deal of energy to better understand cytokine messaging and how signals travel through the body, as well as the role they play in combating various diseases.
According to research, there may be promising applications for cytokines in the diagnosis and treatment of a variety of disorders like cancer, autoimmune diseases, and even septic shock. For scientists to effectively measure cytokine levels in different conditions they need reliable methods. Here we will review cytokines and their clinical uses, as well as the challenges that face cytokine-based research.
Cytokines as Biomarkers and Their Clinical Uses
Cytokine testing comes with its own set of challenges, but its lack of invasiveness and low cost make it an appealing option for researchers. One area of interest where cytokine analysis could be quite helpful is in the battle against chronic inflammation, which has been found to contribute to a whole host of diseases and ailments.
The National Institutes of Health (NIH) weighs in on chronic inflammation stating, “One of the most important medical discoveries of the past two decades has been that the immune system and inflammatory processes are involved in not just a few select disorders, but a wide variety of mental and physical health problems that dominate present-day morbidity and mortality worldwide.”
It turns out that cytokines, including interferons, interleukins and chemokines, all possess an assortment of pro-inflammatory and anti-inflammatory effects throughout the body through biochemical interactions. In recent years, cytokines have been heavily researched to find patterns in certain diseases.
Despite these revelations, utilizing cytokines as biomarkers to research diseases and disorders was not commonplace until recently. In August 2020, researchers at Mount Sinai Health System identified cytokine protein inflammation markers that seemed to predict and measure the severity of COVID-19 in patients as well as predict their survival rate.
A press release from Mount Sinai states, “Researchers studied four proteins known as cytokines that circulate in blood and are commonly associated with infections, and found that two of them, called IL-6 and TNF-α, were able to predict which patients were likely to develop more severe forms of COVID-19 and die.” They added, “This study suggests that these cytokines should be monitored in the treatment of COVID-19 patients to help select those who should enter clinical trials and receive specific drugs that can target them, the researchers say.”
The challenges facing cytokine diagnostics exist in the ability to accurately establish ‘normal’ vs. ‘abnormal’ cytokine levels. Variables differ greatly among individuals and can alter the way cytokines are activated and released into the system. This could be anything from increased stress levels, physiological factors, how physically fit someone is or even dietary habits.
As the accuracy of cytokine testing increases, so does the understanding of how cytokine levels change over time in healthy patients. It also allows researchers the ability to detect health abnormalities earlier in their lifecycle. With enhanced assay sensitivity becoming more readily available, clinicians are finally gaining the tools they need to better prevent diseases through comprehensive cytokine analysis.
Cytokine Platforms and Analysis
Immunoassays currently are the most popular method for determination of cytokines. There are several methods for analyzing cytokines in a sample, with no single method being better than the other. Understanding the different platforms will allow you to decide which one works best for your particular situation and application.
- ELISA assay – The most commonly used form of immunoassay is the enzyme-linked immunosorbent assay (ELISA). This assay uses a primary antibody for the capture, and a secondary antibody conjugated to an enzyme or radioisotope for detection. While the majority of ELISA kits only detect one cytokine at a time, newer multiplexed systems have managed to overcome this limitation to simultaneously detect multiple cytokine expressions in one assay.
- Flow Cytometry (FACS Analysis) – Flow cytometry, commonly referred to as FACS analysis, is a method that proves very accurate and effective when used to identify and measure cellular biomarkers in complex subpopulations. Flow cytometry is used for intracellular cytokine detection and can be conducted in under two hours. This platform can use blood or other bodily fluids such as synovial or cerebrospinal fluid as the specimen for cytokine analysis. Flow cytometry platforms utilize a sophisticated array of lasers, optics, fluidics and electronic detectors to measure light scatter and/or fluorescence emission from cells, running them one at a time through a laser to quantitate physical properties of the cells, including cell size, granularity, and the amounts of target proteins.
- ELISpot – The cytokine ELISpot is both a quantitative and qualitative assay, and has become an important tool for researchers and clinicians to monitor human immunity. This method is commonly used to detect and monitor cellular immune responses to specific antigens, and can assess cytokine release antigen-specific cells and cytokine secretion from activated innate immune cells. The ELISpot assay is based on the enzyme-linked immunosorbent technique and can measure the response of a variety of cell types to a stimulus.
- MSD-ECL – The Meso Scale Discovery platform (MSD-ECL) is similar to the traditional ELISA method in some respects, only the MSD-ECL platform uses non-radioactive electrochemiluminescent labels that are conjugated to detect antibodies. These immunoassays provide ultrasensitive detection, yielding higher sensitivity and broader dynamic range, all at lower sample volumes. MSD platform is suitable for identifying and analyzing trends in multiple cytokine profiles with better precision. The scientists at BioAgilytix are proven experts at multiplexing on the Meso Scale Discovery platform (MSD-ECL). BioAgilytix’s Durham, NC lab was the first to validate MSD’s Human Cytokine 30-Plex V-PLEX Kit, which makes them leaders in MSD-ECL cytokine analysis.
Leading Large-Molecule Bioassay & Immunoassay Lab at BioAgilytix
BioAgilytix is the world’s leading large-molecule bioassay lab. We provide the specialized large molecule insight and proven GLP / GMP knowledge needed to support a full range of cell-based assay requirements. Our veteran team possesses expertise with primary cells and transformed cell lines. Working with our customers, we are able to develop and validate custom cell-based assays in line with all relevant regulatory guidelines and recommendations.
BioAgilytix is the clear choice for cell-based assay and immunoassay development and analysis. We provide accuracy, reliability and world-class expertise. Contact BioAgilytix today for more information on our wide range of bioanalysis services.
