Comparison of Commercial Suppliers of Bead-Based Quantitative Multiplex Cytokine Assays

Introduction

Cytokines are a large group of small signaling molecules that function extensively in cellular communication. This family of proteins consists of immune modulating molecules such as interleukins, chemokines, and interferons; however, they play critical roles in other biological processes as well. Numerous cytokines have been established as biomarkers for diagnosing and treating disease, thus the increasing importance of understanding the roles of these molecules in disease onset and progression, as well as therapeutic targets. Recent research has led to the development of disease-specific panels more accurately assess a variety of diseases, including cardiovascular disease, asthma, inflammation, cancer, diabetes, and rheumatoid arthritis.

The objective of this study was to determine the accuracy of various commercially supplied Luminex xMAP-based multiplex immunoassays in quantitating secreted cytokine levels using stimulated human peripheral blood mononuclear cells (PMBCs) or recombinant standards diluted in normal serum to determine the sensitivity, dynamic range, and scalability of multiplexing experiments. Traditional sandwich ELISAs were used as gold standard references to determine the baseline for cytokine quantitation. Typical use of multiplex immunoassays begin with assessment of a broad range of secreted biomarkers before the number of analytes examined is pared down and the sample number is increased. Therefore, the accuracy of scaling down from 35 to ~12 plexes was also examined by comparing the same analytes across assay kits from different suppliers.

Cytokines

Early cytokine research focused primarily on those related to T helper 1 (TH1) and T helper 2 (TH2) cells, which mediate the cellular and humoral immune responses, respectively. These lymphocytes differentiate from TH0 cells depending on the cytokines present in the cellular environment. TH1 cells arise in response to IFNγ and IL-12 (p70) to play an important role in host defense against bacterial and viral infection. These cells are the primary source of inflammatory cytokines (e.g., lFNγ, IL-2, and TNFβ (LTα)) that stimulate other immune cells to eliminate bacterial pathogens. These cytokines also promote the development of cytotoxic lymphocytes responsible for cell-mediated immunity against viruses and tumor cells. Due to their central role in the immune system, misdirected or hyper-activation of TH1 cells can result in such autoimmune diseases as multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and delayed-type hypersensitivity responses.

Alternatively, TH2 cells mediate the humoral immune response by producing a different variety of cytokines, including IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25). Some of these counteract TH1 responses. Functionally, TH2 cells stimulate and recruit specialized immune cell subsets to sites of infection or in response to allergens or toxins. Like TH1 cells, hyper-activation of TH2 cells can lead to disease, including autoimmune disorders such as chronic graft-versus-host disease, progressive systemic sclerosis, and systemic lupus erythematosus. Additionally, deregulated TH2 responses have been implicated in the development of asthma and other allergic inflammatory diseases and reactions.

TH1 IL-12RB2

IL-18RICOS

CD94

CD19

5

CD26

CD4

NOTCH3

FasL

Tim3

CD254 (RANKL)

TCR

IL-2IFNγTNFαTNFß

T-betRUNX3STAT1STAT4

TH2 CD30

CCR8

TCR

CD4

IL-17RB

NOTCH2

NOTCH1

Tim1

CXCR4

IL-1IL-3IL-4IL-5IL-6

IL-10IL-13

IL-17E/IL-25IL-31

GM-CSF

Gata3STAT6

TH0

IL-27 (early)IL-12p70 (+IL-15) (m)

RANKL

IL-2IL-4

IL-15 (h)IL-17E/IL-25

IL-31IL-33

Current research has moved beyond TH1/TH2 cytokine profiling to now encompass TH9, TH17, TH22, TFH, and Treg cells. Moreover, other non-T cell-associated cytokines are under investigation as well. A better understanding of these cytokines will help further characterize the mechanisms underlying immunity and disease at the cellular, organ, and organismal levels. Such critical information can be obtained from quantitative analysis of cell-specific cytokine profiles. Thus, eBioscience has developed ProcartaPlex panels and Simplex bead sets to easily and efficiently profile these cytokines.

Results

Having developed approximately 1,000 different ELISAs over the past 14 years, eBioscience has established a core competency in superior immunoassay innovation that includes both monoclonal antibody development and their implementation in immunoassays. The ProcartaPlex product line is developed and manufactured with the aim of producing multiplex assays that yield the same sensitivity, linearity, and dynamic range as that of traditional plate-based ELISAs. To validate this objective and determine how ProcartaPlex compares to other commercially available Luminex xMAP-based assay kits, each ProcartaPlex bead set was evaluated alongside a traditional ELISA and three other commercial Luminex-based multiplex panels that contained as many of the same TH1, TH2, and TH17-related analytes as possible.

Regression analysis comparing ProcartaPlex versus sandwich ELISA

Data in figure 1 shows the results of an experiment analyzing six widely studied analytes, namely IFNγ, IL-1β, IL-6, IL-10, IL-17A, and TNFα. Stimulated human (Figure 1a) and mouse (Figure 1b) PBMCs were serially diluted 2-fold in normal human or mouse serum, respectively, to allow for differential cytokine levels in a serum matrix. Regression analysis was performed to determine the correlation between the two different assay types. Data exhibiting an R2 value of at least 0.9 was considered correlative with an R2 value of 1 representing 100% correlation. Our data demonstrate that equivalent results (i.e., R2 > 0.9) were obtained using the ProcartaPlex multiplex immunoassays and traditional plate-based ELISAs for all six human (figure 1a) and mouse (figure 1b) targets, suggesting a high degree of correlation between these two platforms. These results establish ProcartaPlex as a quantitative immunoassay with general assay characteristics comparable to traditional plate-based sandwich ELISAs. Thus, the reduced sample volume, assay time, and cost associated with ProcartaPlex does not come at the expense of assay performance.

TH0

TH9

CD4

TCR

IL-9

TGFßIL-4

TH17IL-23R

IL-13Rα1

IL-21R

IL-1R

1

IL-12RB1

TCR

CD4

CD161

CCR6

IL-17AIL-17F

IL-17AFIL-21IL-22IL-24IL-26

MIP-3α (CCL20)GM-CSF

TNFα

RUNX1BatfIRF4

RORγtRORαSTAT3

AhRc-maf

IL-1ßTGFß+IL-6

IL-21IL-23

IL-2IL-4

IL-27IFNγ

TH22PDGFR

TCR

CCR1

0

CD4

IL-13IL-22FGF

TNFα

TFH CD84

CD4

IL-21R

ICOS

IL-6R

TCR

CD132

CXCR5

gp130

IL-2IL-4

IL-10IL-12IL-21IFNγ

BCL-6STAT3

IL-6IL-21

B Cell Interaction

CXCL13 TReg FR4 (m)

CD39

CD101

CD4

CD73

CD25

GARP (activated)GITR/AITRTCR

CTLA-4

CCR9

CCR4

CXCR3

TGFßIL-10IL-35

Foxp3AhR

STAT5

IL-2TGFβ

TNFαIL-6

Figure 1a

Human IL-6 ProcartaPlex/Platinum ELISA Correlation

Human IL-10 ProcartaPlex/Platinum ELISA Correlation

Human IL-1β ProcartaPlex/Platinum ELISA Correlation Human TNFα ProcartaPlex/Platinum ELISA Correlation

Human IL-17A ProcartaPlex/Platinum ELISA Correlation

Human IFNy ProcartaPlex/Platinum ELISA CorrelationPr

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Figure 1b

Mouse IL-6 ProcartaPlex/Platinum ELISA CorrelationPr

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Mouse IL-1β ProcartaPlex/Platinum ELISA Correlation Mouse TNFα ProcartaPlex/Platinum ELISA Correlation

Mouse IL-17 ProcartaPlex/Platinum ELISA Correlation

Mouse IFNy ProcartaPlex/Platinum ELISA Correlation

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Comparison of ProcartaPlex sensitivity to other commercially available Luminex-based immunoassays

To further characterize ProcartaPlex beyond its quantitative performance relative to traditional ELISAs, this new platform was compared against assay kits from three other commercially available Luminex-based multiplex immunoassay suppliers. In addition to regression analysis between the various suppliers, analyte sensitivity and scalability were also examined. The latter represents each assay’s ability to produce similar results for a particular analyte regardless of whether it is assessed in a 35-plex or a 10-plex panel kit. For comparison, the same analytes evaluated above were used in these multiplex comparisons. Once again, stimulated PBMCs were serially diluted 2-fold in normal human serum.

However, prior to conducting these experiments, the LOD levels of both ProcartaPlex and supplier M were compared in quantitation of 30 human and 25 mouse analytes based on previously published data (Figure 2). The sensitivity trends of the different analytes was similar between the two suppliers with ProcartaPlex consistently showing lower sensitivity levels for all 30 human and 25 mouse analytes. Similar experiments were run in parallel to substantiate these findings as well as their R2 values.

Our comparative analysis demonstrated good correlation between suppliers M and B with the eBioscience ProcartaPlex assay for human IFNγ (Figure 3a). Suppliers M and B performed similarly over a broad range with Supplier M showing nearly identical results to ProcartaPlex, suggesting significantly higher correlation with ProcartaPlex. Data for supplier L is not shown in Figure 3a as the R2 value was too low to be correlative.

To determine the analyte sensitivity of these assays, serially diluted recombinant standards were quantitated. Data in figure 3b shows that, although the assays from the various suppliers performed similarly across the entire assay range, the lower limits of detection (LOD) of the various commercial platforms differed greatly. Results from Supplier M were highly dependent upon the complexity of the panel in which the analyte was incorporated (e.g., 0.30 pg/mL for a 28-plex assay versus 1.60 pg/mL for a 17-plex assay). In the 28-plex panel from supplier M, the LOD of IFNγ was similar to that of ProcartaPlex (0.30 versus 0.27 pg/mL, respectively). However, the 17-plex panel from Supplier M yielded a dramatically higher LOD compared to ProcartaPlex (1.60 versus 0.27 pg/mL, respectively). It is important to note that, although both assays from supplier M were run in parallel using the same samples, a 6-fold difference in LOD was obtained between the two panels. Interestingly, despite exhibiting an R2 value of 0.9131 compared to ProcartaPlex, the LOD of supplier B was 20X greater than that of the eBioscience ProcartaPlex assay. Finally, the assays from Supplier L produced a respectable LOD of 0.60 pg/mL. However, as discussed above, the regression analysis suggested that this assay performed very differently from the other panels tested (including the traditional ELISA), thereby calling into question the accuracy of this panel. This data further substantiates the published data provided in Figure 2.

Human Assay Sensitivities

eBioscience ProcartaPlexSupplier M

eBioscience ProcartaPlexSupplier M

Mouse Assay Sensitivities

Figure 2

Besides human IFNγ, human IL-17A (IL-17) was also examined, albeit to slightly different results. Assays from all three suppliers showed a high correlation (R2 > 0.99) with the eBioscience ProcartaPlex assay, but dramatically different LOD levels (Figure 4). These data illustrate that all four suppliers can produce similar results over the large dynamic range tested. Nevertheless, differences arose in the lower LOD ranges, with ProcartaPlex exhibiting the lowest LOD of all assays tested except the 28-plex from Supplier M. Both ProcartaPlex and supplier M exhibited similar LOD with values of approximately 0.5 pg/mL. However, supplier B yielded a 10X greater LOD than ProcartaPlex.

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The corresponding mouse assays were also evaluated in this study. Although the ProcartaPlex data showed high correlation with commercial ELISA kits, it exhibited a lower R2 value when compared to other commercially available multiplex assays. Figure 5 illustrates that the other suppliers’ kits for quantitating mouse IFNγ and TNFα were less sensitive than ProcartaPlex, as evidenced by the higher LODs observed. Like the human studies, these assays were performed according to the supplier’s instructions using recombinant standards diluted in normal mouse serum. Both ProcartaPlex cytokine panel kits demonstrated consistently low LOD values of approximately 0.30 for IFNγ and 0.28 for TNFα. In the case of IFNγ, suppliers B and L were 4X less sensitive than ProcartaPlex while supplier M was 12X less sensitive. The greatest difference in sensitivity was observed with TNFα as supplier L exhibited an LOD value over 30X that of ProcartaPlex. The reduced sensitivity of the other supplier’s multiplex assays would likely hinder the detection of low level changes of key analytes such as IFNγ and TNFα. This observed lack of sensitivity in the other commercially available multiplex assays is not surprising considering their low correlation with the traditional ELISA and ProcartaPlex assays. Interestingly, no significant correlation was seen amongst the other three suppliers for these mouse targets. They all produced low R2 values between one another. Since the eBioscience ProcartaPlex assay was the only one that demonstrated good correlation with ELISA, it can be reasoned that these kits are the most accurate for quantitating analytes across a large dynamic range that includes extremely low levels of protein.

Figure 4a

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Comparison of multiplex scalability between ProcartaPlex to other commercially available Luminex-based immunoassays

The ability to scale the number of analytes investigated in multiplex experiments is critical for the progression of many projects. For example, it is not uncommon to begin a project by analyzing a large, comprehensive 35-plex cytokine panel on fewer samples (e.g., 100) to determine which analytes are affected by a particular disease or drug treatment. The resultant list of analytes can then be investigated further - for instance, using an 11-plex panel – on a larger set of samples. Thus, there is a great need for scalability in multiplex panel assays in order to enable researchers to correlate all their data across the different stages of investigation. Many commercially available multiplex assays have not been developed with this level consistency in mind. However, this specification is a key design feature that is considered and incorporated during ProcartaPlex assay development.

The data in figure 6 illustrates the high correlation between large- and small-scale multiplex scaling when using the ProcartaPlex assays. Regression analysis comparing the ProcartaPlex Mouse Cytokine & Chemokine Panel 1 (26 plex) and the ProcartaPlex Mouse Th1/Th2 cytokine panel (11 plex) revealed an R2 value > 0.9995 (Figure 6a). This consistency in scalability was also observed in an experiment that quantitated serum IL-6 levels in 18 different human diseased serum samples using the Human Cytokine & Chemokine Panel 1A (34 plex) and Human Th1/Th2 Cytokine Panel (11 plex) assays (Figure 6b). Similar results were obtained when other human and mouse analytes, including IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-13, GM-CSF, IL-12 p70, MCP-1, and TNFα, were quantitated using ProcartaPlex assays. However, when a similar experiment was performed to quantitate human TNFα using supplier M’s 28- and 19-plex assays, the results were inconsistent, suggesting that these assays may not produce reliable, correlative data when scaled (Figure 7).

Supplier M 28 plex

Supplier M 19 plex

Figure 6a

Figure 7

Figure 6b

Human TNFα Quantitation

Discussion and Conclusion

Multiplex immunoassays have proven to be an invaluable tool for comprehensive studies aimed at understanding the complexity of changes observed in secreted cytokine, chemokine, and growth factor abundance. These assays constitute an efficient method for profiling of a large set of biomarkers from a small sample. Many unknowns still remain regarding the biomarker profiles of various diseases and drug treatments. To increase our knowledge, and thus ultimately improve the quality of therapeutic treatments, a multiplex assay platform that reliably maintains sensitivity with scalability is essential. As this paper demonstrates, the eBioscience ProcartaPlex multiplex immunoassays provide a reliable method for quantitating multiple analytes simultaneously with minimal time, cost, and sample volume. The accuracy of these assays has been confirmed against commercially supplied traditional plate-based ELISAs, with which ProcartaPlex assays exhibit a high correlation. Furthermore, unlike other commercially-available multiplex panels, ProcartaPlex assays produce consistent, highly correlative data when scaled to include more or less analytes. Thus, whether the analyte is examined as part of a broad 35-plex assay or a smaller, well-defined T helper cytokine profile, ProcartaPlex assays can detect very low levels of expression with reliability.

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Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334; 6,140,044; 6,399,365; 6,420,169; 6,551,817; 6,733,977; 7,629,164; 7,790,389 and D430,024 and other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637 and 6,054,270. Cyanine (Cy) dye conjugates are covered by US Patent Nos. US5,569,587 and US5,627,027.