Introduction to Multiplex Cytokine Analysis Service

Cytokines are soluble peptides, proteins, and glycoproteins that modulate the immune system. They are crucial for intercellular communication, carrying signals from one cell to another, and modulating immune responses and cell growth. Cytokines are of different types, including chemokines, interleukins, interferons, and lymphokines. Although different cytokines cannot cross the lipid bilayer, they play a critical role in autocrine, endocrine, and paracrine signaling.

Cytokines and chemokines are secreted primarily by the immune cells. Their main objective is to coordinate, proliferate and differentiate cell growth. However, cytokines also participate in numerous other functions, such as chemokines and inflammation, innate and adaptive immunity, angiogenesis, immune cell differentiation, neurobiology, tumorigenesis, viral pathogenesis, and many more.

Dysregulation in cytokine expression is the primary cause of inflammatory and immunological diseases. Several cell types secrete chemokines, often for attracting immune cells at the site of injury or infection, and initiate innate and adaptive responses. As cytokines are at the center of numerous vital body functions, cytokine analysis has received immense effort from the scientific community. Today the focus is on developing cytokine assays such as cytokine detection MSD ELISA to better understand cytokine signaling and decode underlying biological mechanisms.

Current research has shown promising data for cytokine analysis in diagnosing and treating several diseases. Besides, cytokine research has helped understand the Sars-CoV-2 cytokine storm during the Covid-19 pandemic. However, these cytokines need robust bioanalytical techniques for assessing endogenous and treatment regulated levels in complex study matrices. Bioanalytical methods for assessing cytokines range from singleplex ELISA assays to microplate multiplex and MSD assays. The choice of assay of cytokine must fit-for-purpose in the context-of-use.

The current article focuses on the importance of cytokine detection and analysis using Meso Scale Discovery assays. We shall first understand the clinical value of cytokine analysis and then assess different challenges and assay platforms before diving deep into MSD analysis.

Clinical Importance Of Cytokines

Although cytokines and chemokines are crucial in the inflammatory response, testing these cytokines has unique challenges. Chronic inflammation is one frontier where cytokine chemokine can prove helpful and contribute to analyzing a host of ailments and diseases.

The National Institute of Health also agrees with cytokine-chemokine analysis in chronic inflammation. They state that the discovery of the involvement of the immune system in numerous physical and mental issues dominating mortality and morbidity is one of the most crucial revelations of the past two decades. Inflammation cytokines, including chemokines, interferons, and interleukins, possess numerous anti-inflammatory and pro-inflammatory effects. Furthermore, recent research links cytokine patterns with many diseases and disorders.

Despite these discoveries, using inflammation cytokines as biomarkers was still not employed in research on disease and disorders. During the Covid-19 pandemic, researchers at Mount Sinai Health System discovered inflammatory markers that could predict the severity of the infection in patients and predict their rate of survival. The study evaluated four circulatory type of cytokines that were commonly associated with Covid-19 infection. Two of these four proteins, TNF-α and IL-6, were able to predict the severity of Covid-19 infection. These two proteins were able to identify the patients that may develop severe forms of the infection and may die. Hence, this study suggested that one must monitor different cytokines while treating Covid-19 patients.

The major challenge in assessing cytokine analysis is to recognize normal vs. abnormal cytokine levels. Cytokines are influenced by several variables and differ greatly between individuals. Everyone will have a unique way of activating and releasing different types of cytokines into the system. These variables include physiological factors, increased stress levels, dietary habits, and physical fitness.

Cytokines are now widely accepted as biomarkers for multiple metabolic changes and disease states. Detecting and identifying cytokines thus becomes a logical first step in deciphering the underlying biology. Understanding the relationship between cytokines and disease prognosis is the key to developing effective therapeutics.

Multiplex detection of these biomarkers can dramatically accelerate and prove to be a decisive factor in therapeutic strategies.This need has given rise to the use of multiplex cytokine technologies.

With an increase in the accuracy of cytokine testing, the understanding of changes in cytokine levels will also increase over time. This understanding will help detect abnormalities and adopt necessary measures early . Further, with the discovery of more sensitive tools, clinicians and healthcare providers are gaining better insights into managing and preventing diseases through robust cytokine analysis method.

Challenges In Cytokine Detection Methods

As serum and plasma are in close contact with most organs and tissues, they are used widely in cytokine detection method and measurement. The composition of serum and plasma is reflective of the overall functioning of the body. Besides, more tissue-specific fluids, such as CSF and synovial fluids, may offer valuable insights into the local area. Also, tissue lysates and cell culture supernatants are frequently used in cytokine detection assays. However, working with plasma and serum is more suitable as other biological samples are inherently complex. These complex samples contain high levels of collagen, lipids, and other structural proteins that may generate high background signals. Along with masking assay signals, high background noise affects assay validation efforts.

The biological relevance of results is another crucial challenge when detecting and quantifying human cytokines via a human multiplex cytokine assay. Although well-characterized reagents are available for multiplex ELISA cytokine assays, these reagents are often recombinant or synthetic. Besides, most final concentrations of analyte-specific and test-specific reagents do not correlate with each other because of the lack of standardized and well-qualified design materials in assay development. Moreover, the complications rise as most cytokines is sensitive to processing and sample handling protocols. On the other hand, some cytokines, such as IL-12, are present in multiple isoforms, further requiring extensive assay validation. Not to mention, finding a universal dilution factor for both low and high-abundant cytokines is difficult during ELISA multiplex cytokines analysis.

Profiling cytokine release can be problematic, especially in response to an infection. Cytokines not only mediate locally and throughout the body, but they are also pleiotropic, meaning they have multiple functions, and many cytokines may have a shared purpose. Hence, identifying an individual cytokine release in response to an infection is challenging. However, considering the same aspect of cytokine profiling, cytokine detection methods are crucial for estimating the functional capacity of individual cells. Generally, one may determine cell activation by evaluating transcription factors and specific cell surface receptors. However, this is an indirect approach. By using cytokine multiplex assays to detect functional assay for cytokine measurements, one can identify cells producing the cytokine and gain a deeper understanding of the study population.

Cytokine Platform And Analysis Methods

Currently, immunoassays are the most popular method for cytokine expression assays. However, there are numerous factors to consider before selecting a suitable cytokine assay. These factors include analyte of interest, sample matrices, sensitivity, and assay throughput. Each multiplex assay for cytokine detection has its advantages. Understanding the strengths of different multiplex cytokine assays will aid in selecting an ideal platform for all research applications. The following section explores different multiplex assays such as ELISA multiplex cytokine assay, flow cytometry analysis, ELISpot, antibody array, and MSD assay for cytokine detection.

ELISA cytokine assay

Cytokine ELISA is one of the most commonly used functional assays for cytokine detection. Cytokine assays employ a primary antibody for capturing the analyte and a secondary antibody for detection. This secondary antibody is conjugated with an enzyme.

Though ELISA cytokine assays are employed broadly in biomedical and pharmaceutical studies, they still have several limitations. Firstly, they can detect only one analyte at a given time and thus lack multiplexing abilities. For example, during cytokine ELISA analysis of IL-6, IL-1β, and TNF-α, each cytokine needs an independent ELISA kit. Such demands increase the time, cost, labor, and sample volumes. Lastly, ELISA has several washing steps, and therefore it is not a homogenous technique. Besides, it is also not suitable for high-throughput analysis.

Although most biocompare ELISA detects a single inflammatory cytokine, newer multiplex cytokine analysis has overcome this limitation. Multiplex cytokine detection enables the simultaneous detection of numerous cytokines in a single assay volume. Today, one may employ multiplex ELISA cytokine assays to detect several analytes in a single assay volume. However, multiplex ELISA assays will require adequate development and validation initiatives.

Flow cytometry (FACS) analysis

FACS is another effective method to identify and measure cytokines in complex biological matrices. FACS can be used for intracellular and secreted cytokine analysis. Besides serum and plasma, flow cytometry analysis can include CSF and synovial fluids for cytokine analysis. Flow cytometry employs an array of lasers, fluidics, optics, and electronic detectors to measure fluorescence emission and light scatter from cells. The flow cytometer instrument runs the sample through a laser and quantitates the physical properties such as cell size, surface markers, cytotoxicity and granularity of the cells.

ELISpot

ELISpot is both a qualitative and quantitative cytokine assay. Today it has become an important tool for monitoring human immunity. This method can detect and monitor immune responses, cytokine secretion by activated innate immune cells, and cytokine secretion from antigen-specific cells. ELISpot is based on the principle of enzyme-linked immunosorbent technique. Besides, it measures responses against numerous cell types.

Antibody arrays

Antibody arrays are gaining popularity in cytokine research. For example, a study using a quantitative mouse array with 120 analytes showed that Dkk1 promotes hematopoietic regeneration in mice. Besides, a human cytokine array with 80 analytes was successful in identifying secreted molecules from GPR77+ CD10+ cancer-associated fibroblasts. These molecules contribute to chemoresistance when observed in co-cultured tumor cells. The primary advantage of antibody arrays for cytokine analysis is that they are more economical than conventional ELISA. Although antibody arrays may show reduced sensitivity for some analytes, the amount of data generated from single assays generally negates the disadvantage of reduced sensitivity.

Meso Scale Discovery (MSD) assays

MSD cytokine assays are similar to ELISA assays. However, MSD assays use electrochemiluminescence labels to detect analytes in complex study matrices. MSD provides ultrasensitive detection, a broader dynamic range, and higher sensitivity and works well with complex study samples. Hence, MSD assays are ideal for identifying and quantifying multiple cytokines with enhanced precision. MSD offers numerous single plex and multiplex assays for cytokine and chemokine analysis. Some examples of MSD assays include MSD cytokine panels, MSD mouse cytokine panels, and MSD cytokine kits. We at NorthEast Bioanalytical Laboratory are proven experts at analyzing samples with the MSD platform. The following section talks about the MSD platform in more detail.

MSD is a robust platform for measuring cytokines in complex biological matrices. MSD technology enables the profiling of cytokines and chemokines in inflammation. It combines electrochemiluminescence detection and multi-array technology to provide multiplex functionality and unsurpassed sensitivity.

Electrochemiluminescence detection

In an appropriate chemical environment, an MSD instrument supplies electricity to the electrochemiluminescence labels, which generates a light signal. This reaction forms the basis of all MSD instruments. The signal produced is directly proportional to the amount of analyte present in the sample. Some advantages of electrochemiluminescence detection include higher sensitivity, low background noise, broader dynamic range, greater flexibility, ease of use, unsurpassed quality and performance utilizing small volume of test samples.

Multi-array technology

Multi-array technology employs arrays within wells to bring speed and highly dense data to biological systems. This technology coupled with multi-spot plates helps quantify multiple analytes in a single assay volume. Multi-array plates have arrays within them that offer multiplexing capacities and increased throughput. They are available in both 96 and 384 well plates, with each well containing up to 10 spots.

Multi-array plates conserve samples by multiplexing samples with low sample volumes. These achieve higher speed and sensitivity compared to traditional single plex immunoassays besides, offering a broader dynamic range. Hence, cytokines can be analyzed without requiring multiple dilutions.

Instruments

MSD instruments are accessible for basic research as well as applied sciences.

The two primary MSD instruments are MESO SECTOR S 600MM and MESO QuickPlex SQ 120MM. The MESO SECTOR S 600 MM is an industrial capacity ultra-sensitive plate imager. It is equipped with a low-noise high-speed CCD camera to provide a broader dynamic range, high sensitivity, and rapid read times. On the other hand, MESO QuickPlex SQ 120MM offers greater flexibility. It’s a versatile instrument engineered for low-cost experiments, reliable results, and ease of use. Moreover, these MSD instruments work with an efficiently designed software for acquiring and analyzing assay results.

Immunoassays

MSD assay overcomes several challenges associated with traditional immunoassays while providing superior reliability, faster workflows, and higher accuracy and sensitivity. Moreover, multi-spot plates benefit by quantifying several analytes in a single assay volume.

The majority of MSD assays are based on the Sandwich assay principle. These employ Sulfo-tag labels to provide higher-sensitivities. Besides, MSD also offers the option of converting existing ELISA assays to the MSD platform. MSD offers flexibility in numerous ways. Some examples are as follows.

• Direct binding assays in the presence of a single antibody
• Sandwich immunoassays when an antibody pair is available
• Bridging immunoassays for highly sensitive immunogenicity assessments and achieving the higher free drug tolerance
• Competitive immunoassays when needed.

Thus with a plethora of assay applications, the MSD platform is a perfect option for cytokine analysis.

Inflammation and immunology

Inflammation is the body’s response against infection or foreign substances. In inflammation, WBCs release cytokines to protect the body from any damage. This reaction is the natural response of the immune system. By understanding the immune response in the body, one can assess the mechanism of the proposed therapeutics and monitor its development. MSD offers an extensive list of single plex and multiplex immunoassays to support all inflammation and immunology research needs.

Cytokines and chemokines

Cytokines and chemokines are signaling proteins secreted by the immune cells. They are crucial modulatory of numerous biological functions, including immune cell differentiation, inflammation, tumorigenesis, angiogenesis, neurobiology, development, and viral pathogenesis. MSD has a plethora of assay options for all cytokine and chemokine analyses.

Neuroinflammation

Neuroinflammation is a serious issue characterized by pro-inflammatory cytokine concentration, leukocyte invasion, glial activation, and blood-brain-barrier permeability. Neuroinflammation can be caused by several factors, such as infection, toxic metabolites, trauma, or autoimmunity. Current evidence places neuroinflammation at the center of numerous neurological diseases. Besides, studies point to neuroinflammation as a potential target for intervention in Alzheimer’s and other neurological disorders. MSD provides robust support for neuroinflammation research. They have a reliable assay system to measure neuro-inflammatory biomarkers such as Eotaxin, CRP, and MCP-1.

Intracellular signaling

Intracellular signaling is one of the most crucial components of cell biology. Extracellular messages received at the cell surface must be translated into intracellular signaling. These intracellular signals are then converted into complex signaling cascades that regulate cellular responses such as differentiation, proliferation, secretion, and apoptosis. Intracellular signals are generally triggered by the binding of ligands, such as cytokines or growth factors binding to the cell receptors. Upon binding, the stimulus enters the cell and amplifies the signal through signal transduction cascades. MSD has robust assays supporting the research needs of all intracellular signals. MSD assays can detect and measure these signals, including phosphoproteins, ubiquitinated proteins, and GPCR-ligand binding proteins.

Similar to ELISA assays, MSD has different assay formats, including direct, indirect, competitive, and sandwich assay formats. However, MSD assays have advantages over traditional ELISA assays, such as a broader dynamic range and superior sensitivity. This superiority is predominantly due to the chemiluminescent reaction coupled with a reduced number of washing steps. On the other hand, the high binding capacities of MSD carbon electrodes further provide an expansive dynamic assay range. However, due to its non-homogenous nature, MSD assays are not suitable for high-throughput analysis. Although, they do come in 384-well format. Despite being not able to provide high-throughput screening, MSD assays offer multiplexing capacities, a broader dynamic range, MSD custom assay development, and superior sensitivity, making them ideal for detecting biomolecules, including cytokines, in drug discovery and development.

Leading Cytokine Analysis At NorthEast Bioanalytical Laboratories

NorthEast Bioanalytical Laboratories is one of the leading test facilities in cytokine analysis. We provide specialized support through the development of robust ECL (MSD) assays. The veteran team possesses expertise in assay development and validation. Over time, the scientists at NorthEast Biolabs have successfully developed and validated custom ECL assays for accurate, sensitive, and reliable cytokine analysis.