When it comes to diversity, cytokines give scientists many options to explore. These small proteins—often just 5 kDa to 20 kDa—carry signals from one cell to another, and these messages can call out the immunity troops to battle infection, inflammation and more. Cytokines also come in many forms: peptides, proteins and glycoproteins. That assortment of structures demands flexible methods of analysis, such as the ELISA.

In brief, an ELISA uses an antibody that targets a specific antigen. When the two bind, an ELISA generates some indicator, such as a change in color or a fluorescent or chemiluminescent signal, which can be detected with a spectrophotometer or other device. Typically, scientists run ELISAs in 96- or 384-well plates. As Joshua Magnuson, product manager at Bio-Techne, points out, “The ELISA format, despite being in use for over 40 years, remains a top choice to monitor and characterize cytokines for a number of reasons, starting with accurate quantitation.”

Traditionally, an ELISA provides such accuracy because it is so focused, using antibodies that are specific to one cytokine or particular form.

As Magnuson explains, “The ELISA’s focus on a single cytokine allows the assay developer to deal with binding proteins and ligands without concern for how it might affect other cytokines in the sample.” Moreover, it doesn’t take much equipment to run an ELISA. It can be performed with only a plate reader. In addition, this assay is easy, and it “can be performed by any level of researcher and achieve consistent, reproducible results,” Magnuson says.

As tool providers continue to optimize and develop reagents, scientist can expect to perform more detailed analysis on cytokines. Jeremy Lambert, marketing director of Quanterix also shared “Recent developments in ultra-sensitive single-molecule detection of cytokines has enabled researchers to quantitatively measure cytokines with >1000X improvement in sensitivity.” He further adds that “advancement in detection technologies have taken advantage of improved quality of antibody reagent and specifically antibodies with slower off-rates which tend to perform best with ultra-sensitive assays.”

Signal from a sandwich

Like cytokines, ELISAs come in many forms. The so-called sandwich ELISA is considered a very efficient form of the technology.

A sandwich ELISA uses a layer of capture and detection antibodies, and it analyzes antigens between them.

It makes an antibody-antigen-antibody “sandwich.” 

RayBiotech offers more than 2,000 sandwich-based ELISA kits, and “they are all validated with serum, plasma and cell-culture media,” says Valerie Jones, sales and marketing director at RayBiotech. “This is the largest selection of validated sandwich ELISAs on the market—something we’ve been able to accomplish by investing heavily in antibody-pair development.”

RayBiotech’s SpeedELISA runs the entire sandwich immunoassay in a mere three hours—instead of six or seven—because it requires just one wash and three incubation steps. “In this platform, a capture antibody and an HRP [horseradish peroxidase]-conjugated detection antibody are pre-mixed with the sample, allowing immunocapture of the analyte in solution,” Jones notes. “After immobilization of the immunocomplexes onto the microplate, the wells are then ready to receive the chromogenic substrate.”

Miniaturizing the process

By reducing the size of the parts used in this technology, an ELISA can sometimes produce more information. As an example, Bio-Techne developed its Simple Plex assays to run on ProteinSimple’s Ella instrument. Moreover, this combination of technology and assay uses microfluidics to detect multiple antibodies in one run. “By isolating the detection antibodies of each cytokine within a separate channel,” says Magnuson, “it allows the user to avoid many of the pitfalls of multiplexing.”

Although the Simple Plex technology can analyze more than one cytokine at a time, it doesn’t need to, because it can also be formatted to look for one specific cytokine. Additionally, Magnuson explains, “the technology within the Ella instrument allows for a broader dynamic range, giving incredible precision and reproducibility at less than one picogram per milliliter.”

This platform also aims to further simplify an already easy-to-use assay. It takes less than 10 minutes to set up an assay with this system, and it requires even less expertise than a traditional ELISA, because “the standard curve is factory calibrated and preloaded on a barcode, [and there] is no setup time or wells devoted to a standard curve.”

Multiplexing complex pathways

Another advancement in ELISA technology has been the ability to perform multiplex assays.  Cytokines are involved in diverse array of regulatory pathways often with many different proteins interacting in synergy. According to Fiona Coats, vp of marketing at Meso Scale Diagnostics, oftentimes with cytokines, monitoring a single target won’t provide you with the complete story. She adds “The ability to multiplex enables researchers to generate more data from small-volume samples (e.g., mouse samples or limited volume clinical samples).  You have to understand the varying levels of multiple cytokines to generate a clearer picture of what is happening biologically.” Coats also notes, “It is not just improvements related to antibodies-it is all kit components,” that have advanced ELISA methodologies. Coats shares, “Variability in ELISA kits from lot-to lot has been a major pain point for people studying biomarkers, particularly in long-term studies. As we interrogate more proteins in a multiplex analysis, it is important to be confident in the assay system being used is highly reproducible.” Meso Scale Diagnostics offers its U-PLEX immunoassays for flexible multiplexing as well as it V-PLEX validated immunoassays.

In the lab

When studying cytokines, though, not everyone uses the ELISA approach all the time. For example, David Barrow, director of the University of North Carolina’s cytokine and biomarker analysis facility, says, “We are mostly focused on multiplex immunochemistry these days, only choosing ELISA for analytes that aren’t yet commercially available on multiplex.”

Still, many scientists keep using the ELISA approach. For example, a group of Chinese scientists used ELISAs to study the death of cells in sepsis, which can include the release of cytokines [1]. Sepsis is one of the main killers in hospital intensive care units, according to the U.S. National Institute of General Medical Sciences. In particular, the Chinese scientists studied the sepsis-related death of liver cells in an animal, and the team tracked four cytokines using ELISA kits. The results showed that some cytokine levels increase during acute sepsis, and this can accelerate liver damage.

Applications for ELISA-based analysis of cytokines spreads across a wide range of medical conditions, including neurodegenerative ones. As an example, Yuno Song of Korea’s Gyeongsang National University (Jinju) and his colleagues used a mouse model to study Alzheimer’s disease. The researchers wrote: “Alzheimer’s disease (AD) is a complicated neuronal metabolic dysfunction disease that is associated with the induction of inflammation due to microglia cell activation, a loss of synaptic receptors, and neuronal cell loss, that leads to memory loss due to brain lesions” [2]. Using ELISA assays from eBioscience, these scientists analyzed several cytokines in response to treatment with an extract from centipedegrass, which reduces the production of some of the proteins thought to be involved in this disease. The results revealed that the extract triggered the release of anti-inflammatory cytokines rather than ones that fan the inflammation. Consequently, the ELISA results provide some indication of why this extract might protect mice from some molecular drivers of AD. Studies like these could help scientists develop treatments for this neurodegenerative condition and others.

Despite being an old horse in the stable of molecular biology, ELISAs—old and new—keep helping scientists track cytokines in basic and medical research. Moreover, today’s technology tracks these proteins more easily than ever. 

References

[1] Chen, YL, et al., “Inhibition of hepatic cells pyroptosis attenuates CLP-induced acute liver injury,” Am. J. Transl. Res., 8:5685-5695, 2016. [PMID: 28078039] 

[2] Song, Y, et al., “Maysin and its flavonoid derivative from Centipedegrass attenuates amyloid plaques by inducting humoral immune response with Th2 skewed cytokine response in the Tg (APPswe, PS1dE9) Alzheimer’s mouse model,” PLOS One, 12:e0169509, 2017. [PMID: 28072821] 

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