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Immunology Multiplex Assays: Profile More Immune Biomarkers Faster

Learn how MILLIPLEX® cytokine multiplex kits are used at the Forsyth Institute Multiplex Core in Cambridge, MA in this video.

Immunology Multiplex Assays

Quantifying cytokines and other proteins is important for understanding pathological states such as inflammation, sepsis, and diseases such as autoimmune disease, cardiovascular disease, metabolic syndrome, neurological disorders, and cancer. Our wide-ranging and industry-leading immunology multiplex panel offering includes kits to measure cytokines, chemokines, growth factors, soluble cytokine receptors, checkpoint proteins, complement proteins, autoimmune autoantibodies, tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), sepsis-related proteins, skin biomarkers, and immunoglobulins. Popular analytes are described in Table 1.

Table 1.Select popular analytes of immune biomarkers in MILLIPLEX® multiplex immunology assays. See the full list of analytes and MILLIPLEX® multiplex kits in the latest Analyte Quarterly.

Based on Luminex® xMAP® technology, our MILLIPLEX® multiplex immunology assays enable precise, multiparametric analysis of diseases and underlying processes.

Applications of Multiplexing Immune Biomarkers

There are many applications of multiplexing immune biomarkers. This includes:

Below describes examples of how multiplex immunology assays can be used in immunology studies.

Cytokines, Chemokines, and Growth Factors

Acting at the recognition, activation, or effector phases of an immune response, cytokines modulate the development and functional activities of T cells, B cells, and myeloid cells. Using immunology multiplex assays to quantify cytokines, chemokines, and growth factors helps advance research by providing an understanding of various pathological states related to the immune system. Multiplexing allows researchers to investigate analytes simultaneously, saving time, sample, and resources. This is especially critical to immunology research, where numerous immune biomarkers play a role in different pathways.

Learn more about our latest MILLIPLEX® Human and Non-Human Primate Cytokine/Chemokine/Growth Factor multiplex panels.

Interferons are widely expressed cytokines that have potent antiviral and antiproliferative effects. These cytokines are the first line of defense against viral infections and have important roles in immunosurveillance for malignant cells. Figure 1 shows the parallelism of a selection of interferons that the MILLIPLEX® Human Interferon Multiplex Panel can help analyze.

Graphs of the parallelism (linearity) of dilution of interferons IFNα2, IFNλ2, IFNλ3, and IFNγ with the MILLIPLEX® Human Interferon Multiplex Panel (Cat. No. HIFN-130K)

Figure 1.Parallelism/Linearity of dilution using the MILLIPLEX® Human Interferon Panel. IFNα2, IFNλ2, and IFNλ3 analyses were performed with unspiked samples, and IFNγ analysis was performed with spiked samples. Samples were diluted 1:2 as indicated in the protocol and then further diluted as noted in the figure.


The Complement System

The complement system consists of three pathways.

  • The classical pathway is stimulated by antigen-antibody complexes
  • The alternative pathway spontaneously activates on contact with pathogenic cell surfaces
  • The mannose-binding lectin (MBL) pathway recognizes mannose sugars usually present only on pathogenic cell surfaces.

The ability to assess levels of multiple complement proteins in samples to determine complement profiles enables a more accurate characterization of changes in inflammation signaling and innate immune response. The MILLIPLEX® Human Complement panels can be used to determine levels of 16 key complement proteins in serum and plasma samples. The complement cascade is illustrated in Figure 2, which complement components highlighted to indicate which MILLIPLEX® kit they can be measured with. Figure 3 shows example data using these complement panels.

Diagram of the complement cascade pathway. There are 3 different starting points: classical pathway (immune complexes), mannose-binding lectin pathway (mannose on pathogen surface), and alternative pathway (activating surfaces). The classical pathway shows complement components C1q, C1r, C1s, C4, and C2. Mannose-binding lectin pathway shows MBL, MASP-1, MASP-2, C4, and C2. Alternative pathway shows C3, Factor B, and Factor D. All 3 converge onto C3 convertase. C4b is between the classical pathway and C3 convertase, while Properdin and Factor H, Factor I are between alternative pathway and C3 convertase. After this, C3 either goes to C3a for inflammation or C3b to the C5 convertase. C5 can go to C5a for inflammation or to C5b, C6, C7, C8, and C9. Finally the pathway ends on Membrane Attack Complex (MAC, C5b-9). Highlighted components in Product No. HCMPEX1-19K are C2, MBL, C2, Factor D, C4b, Properdin, Factor I, C5, C5a, and C9. Highlighted components in Product No. HCMP2MAG-19K C1q, C4, C3, Factor B, Factor H, C3, and C3b. Highlighted component in Product No. HC5B9-140K is C5b-9.

Figure 2.The complement cascade with highlighted complement components found in the MILLIPLEX® Human Complement Panels. Complement components in blue are found in Product No. HCMPEX1-19K, components in magenta are found in Product No. HCMP2MAG-19K, and components in green are found in Product No. HC5B9-140K.

Box-and-whisker graphs showing significant differences in complement levels when comparing healthy plasma samples to COVID-19 recovery plasma samples using MILLIPLEX® multiplex human complement panels. 4 graphs of different complement components with sample types on the y-axis and concentration in µg/mL on the x-axis. C1q, C5, and C9 showed significance of p<0.05 while C3 showed significance of p<0.005.

Figure 3.Significant differences in complement levels when comparing healthy plasma samples to COVID-19 recovery plasma samples. *p<0.05, **p<0.005.


MMPs and TIMPs

Matrix metalloproteinases (MMPs), a family of zinc proteases responsible for the breakdown of extracellular matrix (ECM), play a key role in normal physiological processes, such as embryonic development and tissue morphogenesis, tissue and bone remodeling, wound healing, and angiogenesis. These processes rely on MMPs′ role in the cleavage of cell surface receptors, the release of apoptotic ligands, cell proliferation and differentiation, and chemokine activity modulation. Similar in structure, MMPs are synthesized and secreted as inactive pro-enzymes that require proteolytic cleavage for activation. This process can be mediated by serine proteases or other MMPs. An increase in MMP expression occurs in response to a wide range of stimuli, including adhesion molecules, growth factors, cytokines, and hormones. Regulation of MMP activity is controlled primarily by tissue inhibitors of metalloproteinases (TIMPs). Therefore, disruption of the MMP/TIMP balance can result in arthritis, cardiovascular disease, and tumor growth and metastasis.


Immune Response and Sepsis Biomarker Analysis

While an immune response is critical for maintaining homeostasis, a hyper-reaction to infection, whether bacterial, viral, fungal, or parasitic, can result in an uncontrolled inflammatory response called sepsis. This hyper-reaction to infection interrupts homeostasis through an uninhibited inflammatory response, including upregulation of sepsis biomarkers, as well as increased apoptosis of lymphoid organs that leads to immune suppression. Severe sepsis or systemic inflammatory response syndrome (SIRS) can cause septic shock, multiple organ dysfunction syndrome (MODS), and death. The MILLIPLEX® Human Sepsis Panels help you understand the role of inflammatory biomarkers involved in sepsis.


Isotyping Human and Mouse Immunoglobulins

Produced by plasma cells and lymphocytes, immunoglobulins (Igs) are crucially involved in immune response, attaching to antigens and playing a role in their destruction. Igs can be classified by isotype, classes that differ in function and antigen response due to structure variability. Five major isotypes are IgM, IgG, IgA, IgE, and IgD – all found in healthy individuals. Some autoimmune diseases, gastrointestinal conditions, and malignancies are characterized by specific isotype deficiencies or varying concentrations of one or more isotypes. Disease states can range from the absence of one isotype class or subclass to a total deficiency of Ig classes.

Researchers can quantitate Ig classes and subclasses simultaneously with MILLIPLEX® Isotyping Kits. The xMAP® multiplex technology is ideal for measuring levels of these isotypes, not only decreasing the number of assays as well as the amount of sample required, but also greatly reducing the possible inaccuracies that result from performing multiple assays.



Fibrosis and related diseases are responsible for an increasing burden of morbidity and mortality worldwide. The MILLIPLEX® Fibrosis Panel 1 enables researchers to advance the understanding of the complex processes associated with fibrotic diseases, including but not limited to idiopathic pulmonary fibrosis, liver fibrosis, and lung fibrosis. Figure 4 shows some example analyte data from this panel.

Graphs showing serum/plasma concentrations for each analyte in MILLIPLEX® Fibrosis Panel 1 from healthy individuals (n=20), systemic lupus erythematosus (SLE; n=7), scleroderma (n=10), Sjögren’s Syndrome (n=4), rheumatoid arthritis (n=4), cystic fibrosis (n=5), pulmonary fibrosis (n=5), and nonalcoholic steatohepatitis (NASH; n=6).

Figure 4.Serum/plasma concentrations for each analyte in Cat. No. HFIB1-100K from healthy individuals (n=20), systemic lupus erythematosus (SLE; n=7), scleroderma (n=10), Sjögren’s Syndrome (n=4), rheumatoid arthritis (n=4), cystic fibrosis (n=5), pulmonary fibrosis (n=5), and nonalcoholic steatohepatitis (NASH; n=6).

Popular Immunology Kits

Explore our popular MILLIPLEX® immunology kits.


For Research Use Only. Not For Use In Diagnostic Procedures.

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