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The Use of Luminex Assays to Measure Cytokines

  • Saami Khalifian
    Correspondence
    Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Ross Research Building 749D, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
    Affiliations
    Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Giorgio Raimondi
    Affiliations
    Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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  • Gerald Brandacher
    Affiliations
    Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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      INTRODUCTION

      Cytokines are small, soluble peptides or proteins that are produced by a variety of cells that participate in the immune response, including T cells, B cells, macrophages, mast cells, fibroblasts, and stromal cells, and are important immune mediators that constitute the communication network of the immune system. Cytokines regulate the responsiveness and maturation of particular cell populations, modulate the balance between cellular and humoral immune responses, and are important determinants of health and disease (
      • O’Shea J.J.
      • Murray P.J.
      Cytokine signaling modules in inflammatory responses.
      ). The cytokine milieu provides important insights into physiological and pathological processes and can potentially serve as biomarkers for various diseases (
      • Paczesny S.
      • Krijanovski O.I.
      • Braun T.M.
      • et al.
      A biomarker panel for acute graft-versus-host disease.
      ;
      • Suh C.H.
      • Kim H.A.
      Cytokines and their receptors as biomarkers of systemic lupus erythematosus.
      ). Therefore, determining the relative balance of cytokines is important in elucidating the biological pathways that lead to inflammation, sepsis, and disease because dysregulation of these complex signaling pathways is often the earliest marker of a pathologic response (
      • Bozza F.A.
      • Salluh J.I.
      • Japiassu A.M.
      • et al.
      Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis.
      ;
      • Chun H.Y.
      • Chung J.W.
      • Kim H.A.
      • et al.
      Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus.
      ).
      In recent years, the Luminex assay has become an important tool in cytokine detection and quantification because of its capacity to measure multiple different cytokines simultaneously in a single run of the assay with small sample size requirements. Thus, this technology enables patterns of numerous cytokines to be examined within and between experiments, which provides a more inclusive and comprehensive depiction of disease than measurement of individual cytokines—an approach that was not previously possible using conventional technologies such as ELISA (
      • Tighe P.
      • Negm O.
      • Todd I.
      • et al.
      Utility, reliability and reproducibility of immunoassay multiplex kits.
      ).

      GENERAL PRINCIPLES AND TECHNIQUE

      Luminex assays enable fast and accurate measurements of cytokines by utilizing hundreds of specially prepared micrometer-scale plastic beads (referred to as microspheres), which are internally dyed with a graded mixture of red or infrared fluorescent dyes (Figure 1A). Varying the degree to which the beads are internally dyed creates hundreds of different fluorescent profiles that can be individually interrogated and classified in a single sample (
      • Tighe P.
      • Negm O.
      • Todd I.
      • et al.
      Utility, reliability and reproducibility of immunoassay multiplex kits.
      ).
      Figure thumbnail gr1
      Figure 1Luminex technique and general principles. (A) Plastic beads (100-μm scale) are internally dyed with a graded mixture of red and infrared fluorescent dyes, providing each microsphere with a unique fluorescent signature. (B) Microspheres of a single identity (i.e., the same fluorescent signature) are conjugated on their surface with a specific capture antibody for a desired cytokine. (C) A mixture of the 100 different microsphere sets is combined with the biological sample of interest in a 96-well plate, incubated, and then washed. (D) Next, a cocktail of detection antibodies conjugated with a reporter dye is added. Notably, a variety of detection antibodies are used, which are specific for different cytokines; however, the reporter dye is the same across all detection antibodies. (E) After another round of washing, the microspheres are analyzed as a single-bead suspension through a flow chamber similar to a flow cytometer. A red laser excites the internal red and infrared fluorescent dyes, which identifies and classifies the bead to one of the 100 microsphere sets. A green laser excites any bound detection antibody–reporter dye complex, and the intensity of this signal quantifies the relative amount of the cytokine bound to the microsphere. (F) Each microsphere traveling through the detection chamber thus has the following components: a capture antibody conjugated to its surface that is specific for a particular cytokine; a variable amount of a specific cytokine that is bound to the capture antibody, which is determined by the concentration of the cytokine in the original sample; and a detection antibody conjugated with a reporter dye that is also specific for the same particular cytokine.
      Figure thumbnail fx1
      The biological sample of interest is then combined with a mixture of microsphere sets—each set specific for a unique cytokine—and incubated in a 96-well plate (Figure 1C). Using a variety of microsphere sets combined within a single assay, it is possible to carry out up to 100 tests in a single well of a 96-well plate. The biological sample–microsphere mixture is then washed, and a cocktail of detection antibodies specific for the desired cytokines is added (Figure 1D). These detection antibodies are also conjugated with a reporter dye, which provides the microsphere with an additional distinct fluorescent emission signature when it binds the cytokine of interest (
      • Tighe P.
      • Negm O.
      • Todd I.
      • et al.
      Utility, reliability and reproducibility of immunoassay multiplex kits.
      ). Thus, it is possible to differentiate various cytokines in a given sample based on the color of the internally dyed beads themselves, while quantifying their relative abundance via measurement of the intensity of fluorescence of the reporter dye–conjugated detection antibody.
      After another round of washing, the microsphere–cytokine–reporter compound is analyzed as a single-bead suspension through a flow chamber equipped with excitation lasers and electronic detection apparatus that measures the intensity of fluorescence of the microsphere–cytokine–reporter compound, similar to a flow cytometer (Figure 1E). Specifically, when a microsphere passes through the detection chamber, a red laser excites both the red and the infrared internal dyes inherent to each particular bead set, which allows the proper classification of the bead to 1 of the 100 microsphere sets. A green laser then excites any reporter dye associated with the binding of the cytokine of interest. Notably, the amount of reporter bound to the microsphere is dependent on the concentration of the particular cytokine in the original biological sample (Figure 1F). The emitted light from the dyes is measured and used for quantitative analysis of each microsphere–cytokine–reporter conjugate.

      APPLICATIONS

      Luminex assays have revolutionized our ability to rapidly assess the relative concentration of various soluble factors that are important biological mediators of health and/or disease. Many rheumatologic, inflammatory, infectious, and genetic conditions are modulated by the differential expression of anti- and proinflammatory cytokines, chemokines, and growth factors, and elucidation of these complex signaling pathways will likely augment our capacity for early diagnosis and drug discovery. For example, Luminex profiling of epidermal explants from patients with systemic sclerosis was recently utilized to identify increased levels of profibrotic CCN2 (a connective tissue growth factor) and proinflammatory protein S100A9 (
      • Nikitorowicz-Buniak J.
      • Shiwen X.
      Abnormally differentiating keratinocytes in the epidermis of systemic sclerosis patients show enhanced secretion of CCN2 and S100A9.
      ). These data suggest that the epidermis of these patients provides an important source of the profibrotic and proinflammatory factors that contribute to the fibrosis and inflammation seen in the disease, and CCN2 and S100A9 emerged as potential markers of disease severity and serve as possible targets for therapeutic intervention.
      The use of Luminex assays for basic science discovery, preclinical/translational studies, and clinical trial immune monitoring has rapidly increased in recent years. In particular, Luminex assays have become widely utilized for biomarker analysis of small-volume samples. In solid-organ and vascularized composite allotransplantation (such as hand and face transplantation), for example, Luminex assays have been utilized to predict high-risk antibodies, positive complement-dependent cytotoxicity crossmatch, and immune monitoring of antibody-mediated rejection and inflammatory cytokines (
      • Campbell P.
      Clinical relevance of human leukocyte antigen antibodies in liver, heart, lung and intestine transplantation.
      ;
      • Schneeberger S.
      • Gorantla V.S.
      • Brandacher G.
      • et al.
      Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.
      ).
      Although the focus of this article is on the use of Luminex assays to quantify relative cytokine expression patterns, the possible applications of this technique extend far beyond cytokines. Indeed, it is possible to conjugate the surface of microspheres with antibodies, receptors, peptides, and oligonucleotides, thereby facilitating performance of immunoassays, receptor–ligand assays, enzyme assays, and DNA assays, among others, which meets the needs of a wide variety of applications including protein expression profiling, gene expression profiling, molecular basis of infectious disease, and HLA testing. In recent years, multiplex PCR and Luminex technology have been used to identify high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among genomic DNA from melanoma-prone families (Figure 2) (
      • Lang J.M.
      • Shennan M.
      • Njauw J.C.
      • et al.
      A flexible multiplex bead-based assay for detecting germline CDKN2A and CDK4 variants in melanoma-prone kindreds.
      ). The Luminex assay quickly and accurately genotyped CDKN2A/CDK4 mutations in a large number of samples, demonstrating its utility as a sensitive, cost-effective, and reliable method for the screening of mutations associated with an increased risk of melanoma. The ability to analyze a variety of biological samples including tissue and cell lysate, saliva, sputum, serum, plasma, and urine adds to the versatility of the Luminex platform as a useful screening modality.
      Figure thumbnail gr2
      Figure 2The use of multiplex PCR and Luminex to screen for genomic variation in familial melanoma. Regions of interest (e.g., CDKN2A) are initially amplified by multiplex PCR. Individual probes with the interrogative terminal codon are linked to a unique 24-mer Tag sequence and subjected to allele-specific primer extension (ASPE). The ASPE extension step uses biotinylated deoxycytidine triphosphates, thereby introducing a biotin-labeled moiety into the extended fragment. The 24-mer Tag/biotinylated ASPE fragment is then specifically captured by a color-coded bead adorned with an anti-Tag, which fully complements the 24-mer Tag sequence. Streptavidin-conjugated phycoerythrin (SA-PE) is then introduced to detect an intensity signal. In summary, the bead color defines the address—i.e., the individual single-nucleotide polymorphism (SNP)—and the PE fluorescence intensity defines the amount of the specific SNP fragment. Reprinted with permission from Lang et al. (2011).

      COMPARISON WITH ELISA AND MICROARRAY FOR CYTOKINE PROFILING

      Luminex has several distinct advantages over similar bioassays, such as ELISA and microarray. Conceptually, the analysis of each microsphere is essentially an ELISA on a bead (
      • Dupont N.C.
      • Wang K.
      • Wadhwa P.D.
      • et al.
      Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants.
      ). This enables multiplexing of up to 100 different analytes in a single well of a Luminex assay because each microsphere serves as an individual test, thereby providing higher throughput than ELISA. The three-dimensional structure and small surface area of the microspheres allow for faster reaction kinetics and shorter assay times than ELISAs and microarrays, which are limited by solid-phase kinetics. Luminex assays also offer the flexibility to customize and attach specific probes to uniquely colored microspheres, easily expanding and readily adapting to specific mutations in specific populations as they are identified, which has important implications for its use as a screening tool. Notably, Luminex requires small sample volumes of only 50 l per well, greatly diminishing the sample volume required compared to ELISA. Thus, more data are generated from the same sample volume.
      Luminex is unique in that it offers both high-density (multiplexing 100 tests per sample) and high-throughput (can test up to 1,000 samples per day) simultaneously, with results that are highly reproducible, reliable, and as accurate as an ELISA. Although ELISA and real-time PCR have the capacity for high throughput (i.e., it is possible to analyze hundreds of samples per day), they are unable to multiplex more than a few tests at a time. Microarrays, on the other hand, provide high-density screening of several hundred tests at a time, but are limited by their lack of reproducibility needed for high-throughput analyses.
      However, several factors may limit the utility and availability of Luminex. First, the Luminex platform requires dedicated analysis instruments that are similar to flow cytometers, which therefore increases the upfront costs associated with this system. Development of newer light-emitting diode–based systems, as opposed to laser-based units, will likely significantly decrease the initial cost to utilize this assay. Luminex assays are also subject to variability because of assay manufacturer, product lot number, and assay execution (
      • Lynch H.E.
      • Sanchez A.M.
      • D’Souza M.P.
      • et al.
      Development and implementation of a proficiency testing program for Luminex bead-based cytokine assays.
      ); however, using the same manufacturer in a given assay and maintaining consistent methodology can minimize variability. Additionally, the presence of human autoantibodies in some biological samples may confound the measurement of cytokines in clinical assays as a result of nonspecific binding. Fortunately, several methods are available to remove these autoantibodies while maintaining the integrity of the resultant cytokine profile (
      • de Jager W.
      • Prakken B.J.
      • Bijlsma J.W.
      • et al.
      Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.
      ).

      CME ACCREDITATION

      This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of the Duke University School of Medicine and Society for Investigative Dermatology. The Duke University School of Medicine is accredited by the ACCME to provide continuing medical education for physicians. To participate in the CME activity, follow the link provided. Physicians should only claim credit commensurate with the extent of their participation in the activity.
      To take the online quiz, follow the link below:

      SUPPLEMENTARY MATERIAL

      A PowerPoint slide presentation appropriate for journal club or other teaching exercises is available at http://dx.doi.org/10.1038/jid.2015.36.

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