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Milliplex assay

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The Milliplex assay is a multiplex immunoassay platform designed for the simultaneous detection and quantification of multiple analytes in a single sample. It utilizes color-coded magnetic beads coated with specific capture antibodies to enable the measurement of various proteins, cytokines, or other biomolecules in a complex biological matrix.

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Lab products found in correlation

Bio plex manager 6, by Bio-Rad (3 mentions) Bio plex device, by Bio-Rad (3 mentions) Xponent software version 4.2 for magpix, by DiaSorin (2 mentions) Elisa kit, by BD (2 mentions) Bio plex apparatus, by Bio-Rad (2 mentions) Anti rat and anti mouse igg magnetic beads, by Qiagen (2 mentions) Magpix luminex platform, by Merck Group (1 mentions) Human cd14 quantikine elisa kit, by R&D Systems (1 mentions) Bio plex 200 system, by Bio-Rad (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Duoset elisa kit, by R&D Systems (1 mentions) Bio plex 200 instrument, by Bio-Rad (1 mentions) Alphalisa, by PerkinElmer (1 mentions) Bio plex suspension array system, by Bio-Rad (1 mentions) Luminex 200 system, by DiaSorin (1 mentions) Colorimetric assay kit, by Fujifilm (1 mentions) A magnetic luminex assay, by R&D Systems (1 mentions) Elisa kit, by R&D Systems (1 mentions) Il 12p70, by BD (1 mentions) Interleukin 6 (il 6), by BD (1 mentions)

30 protocols using milliplex assay

1

Multiplex Analysis of Secreted Biomolecules

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Protein release from OA tissues and stimulated SFs into the CM was evaluated using multiplex bead-based sandwich immunoassay kits. Concentrations of 11 biomolecules (EGF, Eotaxin, FGF-2, IL-10, IL-1Ra, IL-8, IP-10, MCP-1, PDGF-AB/BB, RANTES, VEGF) were quantified at least in duplicate for each sample using MILLIPLEX® Assays (Merck KGaA, Gernsheim, Germany) according to the manufacturer’s protocol and the MAGPIX Luminex platform. Briefly, 25 µL of the standards, controls and samples was added to each well, and then 25 µL of antibody-immobilized beads was added. The plate was incubated overnight at 4 °C, then washed with 200 µL washing buffer and incubated with 25 µL of detection antibodies for 1 h at RT. Finally, the plate was incubated with 25 µL of streptavidin-PE for 30 min at RT, washed and measured in a reader. Next, xPONENT software 4.2 for MAGPIX (Luminex Corporation, Austin, TX) and Bio-Plex Manager 6.1 (Bio-Rad Laboratories, Hercules, CA, USA) were used for data analysis. Once standard curves were generated, concentrations were interpolated for each sample using a 5-parameter curve fitting equation and expressed in pg/mL.
Harvanova D., Matejova J., Slovinska L., Lacko M., Gulova S., Fecskeova L.K., Janockova J., Spakova T, & Rosocha J. (2022). The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis. International Journal of Molecular Sciences, 23(5), 2475.
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2

Multiplex Biomarker Quantification

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Concentrations of 13 biomarkers (EGF, eotaxin, FGF-2, GRO, IL-10, IL-1RA, IL-8, IP-10, MCP-1, PDGF-AB/BB, RANTES, MMP-3, MMP-13) were quantified in duplicate for each sample/patient using MILLIPLEX® Assays (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s protocol and MAGPIX Luminex platform. xPONENT software version 4.2 for MAGPIX (Luminex Corporation, Austin, TX, USA) and Bio-Plex Manager 6.1 (Bio-Rad Laboratories, Hercules, CA, USA) were used for data analysis. Once standard curves were generated, the concentrations for each sample were interpolated using a 5-parameter curve fitting equation and expressed in pg/mL.
Lacko M., Harvanová D., Slovinská L., Matuška M., Balog M., Lacková A., Špaková T, & Rosocha J. (2021). Effect of Intra-Articular Injection of Platelet-Rich Plasma on the Serum Levels of Osteoarthritic Biomarkers in Patients with Unilateral Knee Osteoarthritis. Journal of Clinical Medicine, 10(24), 5801.
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3

Cytokine Profiling of MTEC LPS Exposure

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Cytokines in bronchoalveolar lavage fluid or PBS used in MTEC LPS exposures were measured using Milliplex assays (Millipore, Billerica, MA) according to manufacturer’s instructions.
Laudermilk L.T., Tovar A., Homstad A.K., Thomas J.M., McFadden K.M., Tune M.K., Cowley D.O., Mock J.R., Ideraabdullah F, & Kelada S.N. (2020). Baseline and Innate Immune Response Characterization of a Zfp30 Knockout Mouse Strain. Mammalian genome : official journal of the International Mammalian Genome Society, 31(7-8), 205-214.
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4

Multiplex Cytokine and Chemokine Profiling

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Cryopreserved plasma samples were thawed and mixed by vortexing before assays were performed. Cytokine/Chemokine levels were measured using the Millipore Milliplex® assays (Map Human Cytokine/Chemokine Panel I and IV) and analysed on a BioPlex-200 system (Bio-Rad). The Human Cytokine/Chemokine Panel I included the following cytokines and chemokines: pro-inflammatory [IL-1α, IL-1β, IL-6, IL-12(p40), IL-12(p70), TNFα, IFNα2], chemokines (IL-8, IP-10, MCP-1, MCP-3, MIP1α, MIP1β), adaptive (IFNγ, IL-4, IL-15, IL-17α), anti-inflammatory (IL-10, IL-1RA), and growth factors (VEGF).
The Human Cytokine/Chemokine Panel IV included the following cytokines: IFNβ and IL-28B/IFNλ3. Soluble CD14 (sCD14) levels were measured using the Human CD14 Quantikine® ELISA Kit and human Lipopolysaccharide-Binding Protein (LBP) plasma levels were measured using the LBP kit (R&D Systems Inc, USA). All assays were performed following manufacturer’s instructions. Samples with values outside the range of the standard curve were assigned the value half the limit of detection in pg/mL, LOD/2.
Rambaran S., Naidoo K., Lewis L., Hassan-Moosa R., Govender D., Samsunder N., Scriba T.J., Padayatchi N, & Sivro A. (2021). Effect of Inflammatory Cytokines/Chemokines on Pulmonary Tuberculosis Culture Conversion and Disease Severity in HIV-Infected and -Uninfected Individuals From South Africa. Frontiers in Immunology, 12, 641065.
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5

Multiplex Cytokine Quantification from Serum

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Concentrations of 11 analytes (EGF, FGF-2, IL-10, IL-8, IL-1Ra, IP-10, MCP-1, Eotaxin, PDGF-AB/BB, RANTES, VEGF) were quantified by a triplicate for each serum sample/time point using MILLIPLEX® Assays (Millipore, Merck KGaA, Darmstadt, Germany) to determine the mean of fluorescence intensity (MFI) values according to the manufacturer’s protocol and the MAGPIX Luminex platform. xPONENT software version 4.2 for MAGPIX (Luminex Corporation, Austin, TX, USA) and Bio-Plex Manager 6.1 (Bio-Rad Laboratories, Hercules, CA, USA) were used for data analysis. After creating a standard curve, concentrations were interpolated for each sample and expressed as pg/mL.
Slovinska L., Harvanova D., Janockova J., Matejova J., Cibur P., Moravek M., Spakova T, & Rosocha J. (2022). Mesenchymal Stem Cells in the Treatment of Human Spinal Cord Injury: The Effect on Individual Values of pNF-H, GFAP, S100 Proteins and Selected Growth Factors, Cytokines and Chemokines. Current Issues in Molecular Biology, 44(2), 578-596.
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6

Cytokine Quantification in Lung Homogenates

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Cytokines in the supernatant of lung homogenates were measured by mouse DuoSet ELISA kits (R&D) or Milliplex assays (Millipore) according to manufacturer’s’ instructions. The results were normalized by total protein determined by BCA assay (Thermo Fisher).
Guo X.Z., Dash P., Crawford J.C., Allen E.K., Zamora A.E., Boyd D.F., Duan S., Bajracharya R., Awad W.A., Apiwattanakul N., Vogel P., Kanneganti T.D, & Thomas P.G. (2018). Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity. Immunity, 49(3), 531-544.e6.
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7

Multiplexed Protein Quantification in Bloodspots

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The BBV Core performed multiplexed magnetic fluorescent bead-based immunoassays (Milliplex assays, Millipore) to measure levels of neuroprotective factors (BDNF, IGF-1, FGF-2, VEGF-A) and inflammatory biomarkers (TNFα, CRP, IL-1α, IL-1β, IL-2, IL-6, IFN-γ) in the bloodspot extracts. Quantification was based on standard curves included in each assay run.
These assays essentially are sandwich ELISAs performed on the surface of beads that can be multiplexed due to the fluorescent nature of the beads and thus require much smaller sample volumes than traditional ELISAs performed as single-plex assays. The Milliplex assays were performed following manufacturer’s protocol in batches which included all three timepoints for each patient. Briefly, this involved overnight incubation of the protein extract at 4 °C with the magnetic beads on an orbital shaker (600 rpm) followed by addition of a biotinylated detection antibody and a streptavidin-conjugated phycoerythrin reporter. Media fluorescence intensity (MFI) was measured for all unknown samples and standards using a Bioplex 200 instrument (BioRad). The Bioplex Manager software (v 4.1.1) was used to generate standard curves for each analyte and calculate the analyte concentrations in each unknown sample. Analyte concentrations were normalized to total protein in each sample.
Myers J.S., Pathak H.B., He J., Ghosh A., Puri R.V., Asakura Y, & Miyashita M. (2022). Combined Exercise and Game-based Cognitive Training Intervention: Correlative Pilot Study of Neurotrophic and Inflammatory Biomarkers for Women with Breast Cancer. Cancer nursing.
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8

Quantification of Cytokine Responses

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Supernatants collected 24 h after infection at a MOI of 3, and D0–D5 after infection at a MOI of 0.1, were used to quantify secreted cytokines. IFN-β, RANTES/CCL5, IP-10/CXCL10 and IL-1β were quantified using ALPHAlisa (Perkin Elmer), IFN-λ1 and thymic stromal lymphopoietin (TSLP) were quantified using standard ELISA (eBioScience). IFN-λ2/3, IFN-α, and IFN-γ were quantified by Milliplex assay (Millipore). RNA was extracted from cells infected at a MOI of 3, and RT-qPCR performed to quantify IRF7, Mx1 and IL-33 mRNA (see online supplementary information).
Spann K.M., Baturcam E., Schagen J., Jones C., Straub C.P., Preston F.M., Chen L., Phipps S., Sly P.D, & Fantino E. (2014). Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy. Thorax, 69(10), 918-925.
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9

Quantifying G-CSF Expression in Skeletal Muscle

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A Milliplex assay (Millipore) was used to analyse G-CSF protein expression in the skeletal muscle and plasma samples following the manufacturer's protocol (Millipore). Briefly, the samples and standards were added to a 96 well plate containing premixed beads coated with G-CSF antibody. Following a 30 min incubation plates were washed three times then incubated with pre-mixed detection antibodies. Wells were then washed and incubated with streptavidin-phycoerythrin. After a final series of washes samples were resuspended in 125 μ l of assay buffer. The plate was read on the Bio-Plex Suspension Array System (V.5.0, Bio-Rad). For skeletal muscle, samples were extracted according to the protein extraction method described above and diluted to 1000 μg/ml prior to the assay. Plasma samples were run undiluted. Intra-assay coefficient of variation (CV%) was 4.2% for the plasma and 7.1% for the diaphragm homogenate.
Wright C.R., Brown E.L., Della-Gatta P.A., Ward A.C., Lynch G.S, & Russell A.P. (2014). G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle. Frontiers in Physiology, 5, 170.
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10

Measuring Plasma Cytokine Levels in Postnatal Mice

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Measurement of the levels of cytokines within plasma collected at post-natal day 7 and 120 was performed using Luminex 200 system (Luminex, Austin, Tex.). Plasma concentrations of interleukin (IL) -2, interferon-γ (IFN-γ), IL-4, IL-5, IL-13, and IL-17A were assessed using the Milliplex Assay (Millipore) system. The study method was modified from previously reported methods [24] (link), [25] (link). Antibody conjugated beads were incubated first with diluted standards or plasma from animal subjects for 2 hours and then with detector antibodies for 1 hour at room temperature. Fluorescent detection was performed after the sample had been incubated for 1.5 hours with biotin as reporter and incubated for 30 min with fluorescent dye-conjugated streptavidin-phycoerythrin. Cytokine levels were measured by using a flow cytometer and were analyzed with Flowmetrix software (Master Plex QT 1.2 system) [24] (link).
Kuo H.C., Guo M.M., Liu S.F., Chen C.C., Sheen J.M., Yu H.R., Tiao M.M., Tain Y.L, & Huang L.T. (2014). Cross-Fostering Increases Th1/Th2 Expression in a Prenatal Dexamethasone Exposure Rat Model. PLoS ONE, 9(12), e115554.
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