Comparison of Multiplex Immunoassay Platforms.

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Comparison of Multiplex Immunoassay Platforms.

Fu, Q., Zhu, J., Van Eyk, J.E.

Journal		Clinical Chemistry.
Species
Analytes Measured		IL-10 , IL-12 p70 , IL-2 , IL-6
Matrix Tested		Serum

Year		2010
Volume		56
Page Numbers		314-318
Application		Cytokines and Chemokines

Abstract

BACKGROUND: Candidate biomarkers discovered with high-throughput proteomic techniques (along with many biomarkers reported in the literature) must be rigorously validated. The simultaneous quantitative assessment of multiple potential biomarkers across large cohorts presents a major challenge to the field. Multiplex immunoassays represent a promising solution, with the potential to provide quantitative data via parallel analyses. These assays also require substantially less sample and reagents than the traditional ELISA (which is further limited by its ability to measure only a single antigen). We have measured the reproducibility, reliability, robustness, accuracy, and throughput of commercially available multiplex immunoassays to ascertain their suitability for serum biomarker analysis and validation.

METHODS: Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A(2) (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits.

RESULTS: The quantifiable interval determined for each assay and antigen was based on precision (CV < 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (10(5) to 10(6)). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays.

CONCLUSIONS: The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.

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U-PLEX Human IL-10 384-Well Assay IL-10 \| Human Singleplex
U-PLEX Human IL-6 384-Well Assay IL-6 \| Human Singleplex
U-PLEX Custom Metabolic Group 1 (human) 384-well Assays BAFF, BDNF, β-NGF, C-Peptide, CTACK, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, EPO, FGF-21, FGF-23, FLT3L, Fractalkine, FSH, G-CSF, Ghrelin (active), Ghrelin (total), GIP (active), GIP (inactive), GIP (total), GLP-1 (active), GLP-1 (inactive), GLP-1 (total), Glucagon, GM-CSF, GRO-α, I-309, IFN-α2a, IFN-β, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17C, IL-17D, IL-17E/IL-25, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-27, IL-29/IFN-λ1, IL-31, IL-33, Insulin, IP-10, Leptin, Luteinizing Hormone (LH), MCP-1, MCP-2, MCP-4, M-CSF, MDC, MIF, MIP-1α, MIP-5, PP, Proinsulin, PYY (total), SDF-1α, TARC, TNF-α, TNF-β, TPO, TRAIL, TSLP, VEGF-A, YKL-40 \| Human Multiplex
U-PLEX Custom Biomarker Group 1 (human) 384-well Assays CTACK, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, EPO, FLT3L, Fractalkine, G-CSF, GM-CSF, GRO-α, I-309, IFN-α2a, IFN-β, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-27, IL-29/IFN-λ1, IL-31, IL-33, IP-10, I-TAC, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIF, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-5, SDF-1α, TARC, TNF-α, TNF-β, TPO, TRAIL, TSLP, VEGF-A, YKL-40 \| Human Multiplex
S-PLEX Proinflammatory Panel 1 (human) Kit IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, TNF-α \| Human Multiplex
U-PLEX Macrophage M2 Combo 1 (mouse) IL-4, IL-10, IL-13, MDC, TARC \| Mouse Multiplex
S-PLEX NHP IL-2 Kit IL-2 \| Non-human primate Singleplex
S-PLEX NHP IL-6 Kit IL-6 \| Non-human primate Singleplex
S-PLEX Human IL-6 Kit IL-6 \| Human Singleplex
S-PLEX Human IL-10 Kit IL-10 \| Human Singleplex
S-PLEX Human IL-2 Kit IL-2 \| Human Singleplex
S-PLEX Human IL-12p70 Kit IL-12p70 \| Human Singleplex
U-PLEX CAR-T Cell Combo 1 (human) GM-CSF, Granzyme A, Granzyme B, IFN-γ, IL-2, TNF-α \| Human Multiplex
U-PLEX Macrophage M2 Combo 1 (human) Eotaxin-2, IL-4, IL-10, IL-13, M-CSF, MDC, TARC \| Human Multiplex
U-PLEX Macrophage M1 Combo 1 (human) IL-1β, IL-6, IL-12p70, IL-18, IL-23, IP-10, MCP-1, MIP-1α, TNF-α \| Human Multiplex
V-PLEX Viral Panel 3 NHP Kit IFN-γ, IL-1β, IL-5, IL-6, IL-8, IL-10 \| Non-human primate Multiplex
V-PLEX Viral Panel 3 Human Kit IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α \| Human Multiplex
V-PLEX Plus Viral Panel 3 NHP Kit IFN-γ, IL-1β, IL-5, IL-6, IL-8, IL-10 \| Non-human primate Multiplex
V-PLEX Plus Viral Panel 3 Human Kit IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α \| Human Multiplex
U-PLEX Viral Combo 1 (NHP) G-CSF, GM-CSF, IFN-α2a, IFN-γ, IL-1β, IL-1RA, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IP-10, MCP-1, MIP-1α, TNF-α, VEGF-A \| Non-human primate Multiplex
U-PLEX Viral Combo 1 (human) G-CSF, GM-CSF, IFN-α2a, IFN-β, IFN-γ, IL-1β, IL-1RA, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IP-10, MCP-1, MIP-1α, TNF-α, VEGF-A \| Human Multiplex
V-PLEX Viral Panel 2 NHP Kit IFN-γ, IL-1β, IL-6, IL-8, IL-10 \| Non-human primate Multiplex
V-PLEX Viral Panel 2 Human Kit IFN-γ, IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α \| Human Multiplex
V-PLEX Viral Panel 1 NHP Kit IL-1β, IL-6, IL-8 \| Non-human primate Multiplex
V-PLEX Viral Panel 1 Human Kit IL-1β, IL-6, IL-8, TNF-α \| Human Multiplex
V-PLEX Plus Viral Panel 2 NHP Kit IFN-γ, IL-1β, IL-6, IL-8, IL-10 \| Non-human primate Multiplex
V-PLEX Plus Viral Panel 2 Human Kit IFN-γ, IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α \| Human Multiplex
V-PLEX Plus Viral Panel 1 NHP Kit IL-1β, IL-6, IL-8 \| Non-human primate Multiplex
V-PLEX Plus Viral Panel 1 Human Kit IL-1β, IL-6, IL-8, TNF-α \| Human Multiplex
U-PLEX NHP IL-2 Assay IL-2 \| Non-human primate Singleplex
U-PLEX NHP IL-6 Assay IL-6 \| Non-human primate Singleplex
U-PLEX NHP IL-10 Assay IL-10 \| Non-human primate Singleplex
U-PLEX Mouse IL-10 Assay IL-10 \| Mouse Singleplex
U-PLEX Mouse IL-6 Assay IL-6 \| Mouse Singleplex
U-PLEX Mouse IL-12p70 Assay IL-12p70 \| Mouse Singleplex
U-PLEX Mouse IL-2 Assay IL-2 \| Mouse Singleplex
U-PLEX NHP IL-12p70 Assay IL-12p70 \| Non-human primate Singleplex
U-PLEX Human IL-6 Assay IL-6 \| Human Singleplex
U-PLEX Human IL-12p70 Assay IL-12p70 \| Human Singleplex
U-PLEX Human IL-10 Assay IL-10 \| Human Singleplex
U-PLEX Human IL-2 Assay IL-2 \| Human Singleplex
R-PLEX Rat IL-10 Assay IL-10 \| Rat Singleplex
R-PLEX Rat IL-2 Assay IL-2 \| Rat Singleplex
R-PLEX Rat IL-6 Assay IL-6 \| Rat Singleplex
U-PLEX Custom Metabolic Group 1 (mouse) Assays Mouse Multiplex
U-PLEX Adipokine Combo 1 (mouse) BDNF, IL-1β, IL-6, IL-10, Insulin, Leptin, MCP-1, TNF-α \| Mouse Multiplex
U-PLEX Metabolic Hormones Combo 1 (mouse) C-Peptide, Ghrelin (active), GLP-1 (active), Glucagon, IL-6, Insulin, Leptin, MCP-1, PYY (total), TNF-α \| Mouse Multiplex
U-PLEX Custom Metabolic Group 1 (human) Assays BAFF, BDNF, β-NGF, C-Peptide, CTACK, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, EPO, FGF-21, FGF-23, FLT3L, Fractalkine, FSH, G-CSF, Ghrelin (active), Ghrelin (total), GIP (active), GIP (inactive), GIP (total), GLP-1 (active), GLP-1 (inactive), GLP-1 (total), Glucagon, GM-CSF, GRO-α, I-309, IFN-α2a, IFN-β, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17C, IL-17D, IL-17E/IL-25, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-27, IL-29/IFN-λ1, IL-31, IL-33, Insulin, IP-10, Leptin, Luteinizing Hormone (LH), MCP-1, MCP-2, MCP-4, M-CSF, MDC, MIF, MIP-1α, MIP-5, PP, Proinsulin, PYY (total), SDF-1α, TARC, TNF-α, TNF-β, TPO, TRAIL, TSLP, VEGF-A, YKL-40 \| Human Multiplex
U-PLEX Adipokine Combo 1 (human) BDNF, β-NGF, IL-1β, IL-6, IL-8, IL-10, Insulin, Leptin, MCP-1, TNF-α \| Human Multiplex
R-PLEX Rat IL-6 Antibody Set IL-6 \| Rat
R-PLEX Rat IL-10 Antibody Set IL-10 \| Rat
R-PLEX Rat IL-2 Antibody Set IL-2 \| Rat
V-PLEX Plus Mouse Cytokine 19-Plex Kit IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, IP-10, KC/GRO, MCP-1, MIP-1α, MIP-2, TNF-α \| Mouse Multiplex
V-PLEX Mouse Cytokine 19-Plex Kit IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, IP-10, KC/GRO, MCP-1, MIP-1α, MIP-2, TNF-α \| Mouse Multiplex
U-PLEX T-Cell Combo (NHP) GM-CSF, IFN-γ, IL-2, IL-4, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, MIP-3α, TNF-α \| Non-human primate Multiplex
U-PLEX T-Cell Combo (mouse) GM-CSF, IFN-γ, IL-2, IL-4, IL-9, IL-10, IL-13, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, MIP-3α, TNF-α \| Mouse Multiplex
U-PLEX T-Cell Combo (human) GM-CSF, IFN-γ, IL-2, IL-4, IL-9, IL-10, IL-13, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, MIP-3α, TNF-α \| Human Multiplex
U-PLEX TH17 Combo 2 (mouse) IFN-γ, IL-1β, IL-6, IL-17A, IL-17C, IL-17E/IL-25, IL-17F, IL-21, IL-22, TNF-α \| Mouse Multiplex
U-PLEX NHP IL-12p70 Antibody Set IL-12p70 \| Non-human primate
U-PLEX TH17 Combo 2 (human) IFN-γ, IL-1β, IL-6, IL-10, IL-17A, IL-17E/IL-25, IL-17F, IL-21, IL-22, TNF-α \| Human Multiplex
U-PLEX TH1/TH2 Combo (NHP) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, TNF-α \| Non-human primate Multiplex
U-PLEX TH17 Combo 1 (NHP) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, TNF-α \| Non-human primate Multiplex
U-PLEX Human IL-6 Antibody Set IL-6 \| Human
U-PLEX Human IL-12p70 Antibody Set IL-12p70 \| Human
U-PLEX Human IL-10 Antibody Set IL-10 \| Human
U-PLEX Human IL-2 Antibody Set IL-2 \| Human
U-PLEX Mouse IL-12p70 Antibody Set IL-12p70 \| Mouse
U-PLEX Mouse IL-10 Antibody Set IL-10 \| Mouse
U-PLEX Mouse IL-6 Antibody Set IL-6 \| Mouse
U-PLEX Mouse IL-2 Antibody Set IL-2 \| Mouse
U-PLEX TH1/TH2 Combo (human) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, TNF-α \| Human Multiplex
U-PLEX Proinflam Combo 2 (human) GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 \| Human Multiplex
U-PLEX TH1/TH2 Combo (mouse) IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, KC/GRO, TNF-α \| Mouse Multiplex
U-PLEX Proinflam Combo 1 (NHP) IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12/IL-23p40, TNF-α \| Non-human primate Multiplex
U-PLEX Biomarker Group 1 (NHP) Assays CTACK, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, FLT3L, Fractalkine, G-CSF, GM-CSF, GRO-α, I-309, IFN-α2a, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-17F, IL-18, IL-22, IL-23, IP-10, I-TAC, MCP-1, MCP-2, MCP-4, M-CSF, MDC, MIF, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-5, SDF-1α, TARC, TNF-α, TNF-β, TPO, TRAIL, VEGF-A, YKL-40 \| Non-human primate Multiplex
U-PLEX Custom Biomarker Group 1 (human) Assays CTACK, ENA-78, Eotaxin, Eotaxin-2, Eotaxin-3, EPO, FLT3L, Fractalkine, G-CSF, GM-CSF, GRO-α, I-309, IFN-α2a, IFN-β, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-2Rα, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12/IL-23p40, IL-12p70, IL-13, IL-15, IL-16, IL-17A, IL-17A/F, IL-17B, IL-17C, IL-17D, IL-17E/IL-25, IL-17F, IL-18, IL-21, IL-22, IL-23, IL-27, IL-29/IFN-λ1, IL-31, IL-33, IP-10, I-TAC, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIF, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-5, SDF-1α, TARC, TGF-β1, TGF-β2, TGF-β3, TNF-α, TNF-β, TPO, TRAIL, TSLP, VEGF-A, YKL-40 \| Human Multiplex
U-PLEX Proinflam Combo 1 (human) IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α \| Human Multiplex
U-PLEX Proinflam Combo 4 (human) IL-1β, IL-6, IL-8, TNF-α \| Human Multiplex
U-PLEX Proinflam Combo 3 (human) IFN-γ, IL-1β, IL-6, TNF-α \| Human Multiplex
V-PLEX Proinflammatory Panel 2 Rat Kit IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, KC/GRO, TNF-α \| Rat Multiplex
V-PLEX Plus Proinflammatory Panel 2 Rat Kit IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, KC/GRO, TNF-α \| Rat Multiplex
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Comparison of Multiplex Immunoassay Platforms.

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