Duoset elisa

Manufactured by R&D Systems

Sourced in United States, United Kingdom, Germany, France, Italy, Canada

The DuoSet ELISA is a set of components for the development of sandwich ELISA assays. It includes matched antibody pairs and detection reagents for the measurement of specific proteins.

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635 protocols using duoset elisa

Quantification of Inflammatory Mediators in Murine Models

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Recombinant murine SAP from mouse myeloma cell line NSO was used (lot. #MPX1518061; R&D Systems). Native SAP from human serum was purchased by Merck-Millipore (lot. #2787319). Recombinant murine PTX3 was purified from supernatant of Chinese hamster ovary cells (ATCC^®-CRL-12023) stably expressing the protein, as described previously^{51 (link)}. Purity of the recombinant protein was assessed by SDS-PAGE followed by silver staining. Proteins contained <0.125 endotoxin units/ml as checked by the Limulus amebocyte lysate assay (BioWhittaker^®, Inc.). SAP levels were measured in mouse plasma by ELISA (DuoSet ELISA; R&D Systems) after collection of blood from the cava vein. Murine MPO was measured in BALFs by ELISA (DuoSet ELISA; R&D Systems). Murine CXCL1, CXCL2, TNF-α and IL-6 were measured in supernatants by ELISA (DuoSet ELISA; R&D Systems) according with manufacturer’s instructions. Murine C5a was measured either in plasma-heparin or in BALFs previously stored at −80 °C by DuoSet ELISA (R&D Systems) maintaining EDTA (10 mM) throughout the assay in order to stop the activation of the complement cascade. Human SAP was measured in sera and BALFs of patients by ELISA (lot. #P105304, DuoSet ELISA; R&D Systems) according with manufacturer’s instructions.

Doni A., Parente R., Laface I., Magrini E., Cunha C., Colombo F.S., Lacerda J.F., Campos A J.r., Mapelli S.N., Petroni F., Porte R., Schorn T., Inforzato A., Mercier T., Lagrou K., Maertens J., Lambris J.D., Bottazzi B., Garlanda C., Botto M., Carvalho A, & Mantovani A. (2021). Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus. Nature Communications, 12, 3739.

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Fasting Biomarker Evaluation Protocol

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After an overnight fast (>8 h), blood samples were collected and aliquots of plasma and serum were snap frozen in liquid nitrogen and stored at −80°C. An electrolyte panel, a liver panel, a glycemia panel, a lipid panel, and CRP were analyzed using standardized laboratory techniques as previously described.^{22 (link)} Change in insulin sensitivity between baseline and 6 weeks, as estimated by the HOMA-IR, was the primary outcome (HOMA2 Calculator, University of Oxford).
As previously described, IL-1β, IL-6, IL-10, TNF-α, and LPS were quantified by enzyme-linked immunosorbent assay (ELISA) as part of the systematic inflammation evaluation.^{22 (link)} Appetite and glucose metabolism-regulating hormones (leptin and ghrelin) were measured using a ghrelin assay (R&D Systems DuoSet ELISA, DY8149–05) and a leptin assay (R&D Systems DuoSet ELISA, DY398–05) (R&D Systems DuoSet ELISA, DY398–05).

Zhang Z., Mocanu V., Deehan E.C., Hotte N., Zhu Y., Wei S., Kao D.H., Karmali S., Birch D.W., Walter J, & Madsen K.L. (2024). Recipient microbiome-related features predicting metabolic improvement following fecal microbiota transplantation in adults with severe obesity and metabolic syndrome: a secondary analysis of a phase 2 clinical trial. Gut Microbes, 16(1), 2345134.

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Cytokine Profiling in Myelodysplastic Syndrome

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Peripheral blood plasma from four healthy donors, as well as the same six low/intermediate-risk and six high-risk MDS patients from the CyTOF studies, was stored at −80°C. IL-6 (Human IL-6 DuoSet ELISA), IL-8 (DuoSet ELISA [enzyme-linked immunosorbent assay] Human IL-8/CXCL8), TGFβ (human TGFβ1), and TNFα (Quantikine ELISA human TNFα) were measured according to kit instructions (R&D Systems, Minneapolis, MN) and read at 450 nm with a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA).

Paracatu L.C., Monlish D.A., Greenberg Z.J., Fisher D.A., Walter M.J., Oh S.T, & Schuettpelz L.G. (2022). Toll-like receptor and cytokine expression throughout the bone marrow differs between patients with low- and high-risk myelodysplastic syndromes. Experimental hematology, 110, 47-59.

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Quantifying Bone and Immune Markers in Irradiated Mice

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Peripheral blood (50–500 μL) from irradiated and nonirradiated C57BL/6 mice was regularly collected every several weeks with 1.5 mL microcentrifuge tubes from the submandibular vein with a 0.5‐mm Goldenrod Animal Lancet (MEDIpoint, New York, USA). After the blood was collected, the hemorrhagic area was compressed with KimWipes to stop the bleeding. The collected blood was allowed to clot for at least 30 minutes at room temperature. The tubes were centrifuged at 3000g for 10 minutes at 4°C. Serum was collected and stored at −80°C until use. All measurements were performed with an ELISA kit or DuoSet ELISA kit in accordance with the manufactures' instructions. The serum levels of the bone resorption marker CTX‐I were measured by ELISA (AC‐06F1, IDS, RatLaps Collagen I). The serum levels of the bone formation markers PINP and OPG were measured by ELISA (AC‐33F1, IDS) and DuoSet ELISA (DY459, R&D systems), respectively. The serum levels of the CCL2 cytokine and CXCL1 chemokine were measured by DuoSet ELISA (DY479 for CCL2, DY453 for CXCL1, R&D Systems). A standard curve was generated for each protein, and concentrations were extrapolated from the standard curve.

Kohzaki M., Ootsuyama A., Abe T., Tsukamoto M., Umata T, & Okazaki R. (2022). Long Bones Exhibit Adaptive Responses to Chronic Low‐Dose‐Rate Ionizing Radiation despite its Lifespan‐Shortening and Carcinogenic Effects on C57BL/6 Mice. JBMR Plus, 6(12), e10688.

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Quantifying CXCL10 in Milk Whey

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Human CXCL10/IP-10 DuoSet ELISA (R&D Systems, Minnesota, USA) was used to determine cytokine concentrations in pg/ml. Assays were performed in duplicates according to the manufacturer’s instructions. DuoSet ELISA (R&D Systems, Minnesota, USA) for CXCL10 was adjusted for the measurement of milk whey using 1.75 % nonfat dried milk powder (AppliChem GmbH, Darmstadt, Germany) in phosphate-buffered saline as reagent diluent, which made it possible to measure milk whey samples undiluted with a recovery rate of 99.3 % [range: 73.7 %; 120.0 %] (Table S1). Concentrations were obtained from a standard curve using a 4 Parameter Logistic Curve fit on GraphPad Prism (GraphPad Software inc., Version 8.1.0, San Diego, USA).

Rabe T., Lazar K., Cambronero C., Goelz R, & Hamprecht K. (2020). Human Cytomegalovirus (HCMV) Reactivation in the Mammary Gland Induces a Proinflammatory Cytokine Shift in Breast Milk. Microorganisms, 8(2), 289.

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Intestinal Cytokine Quantification

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Levels of IL-8 and TNF-α in homogenized intestinal tissue were determined with porcine IL-8/CXCL8 DuoSet ELISA (DY535, R&D Systems, Abingdon, UK) and porcine TNF-alpha DuoSet ELISA (DY690B, R&D Systems, Abingdon, UK) according to manufacturer’s instructions, in 50 µL undiluted sample. Cytokine levels were corrected for protein concentration in the samples, as determined by BCA.

Navis M., Muncan V., Sangild P.T., Møller Willumsen L., Koelink P.J., Wildenberg M.E., Abrahamse E., Thymann T., van Elburg R.M, & Renes I.B. (2020). Beneficial Effect of Mildly Pasteurized Whey Protein on Intestinal Integrity and Innate Defense in Preterm and Near-Term Piglets. Nutrients, 12(4), 1125.

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Quantification of Inflammatory Markers

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IL-1β and IL-6 were quantified by enzyme-linked immunosorbent assay (ELISA) (Limit of detection (LOD): 7.8 pg/ml, DuoSet ELISA, R&D Systems, Minneapolis, USA) in BALF following manufacturer’s instructions. HIF-1α (LOD: 31.25 pg/ml, DuoSet ELISA, R&D Systems) was assessed in SN of lung homogenates (centrifuged 10 min at 240 g, 4 °C) or in the cell culture after lysis following manufacturer’s instructions.

Sironval V., Palmai-Pallag M., Vanbever R., Huaux F., Mejia J., Lucas S., Lison D, & van den Brule S. (2019). HIF-1α is a key mediator of the lung inflammatory potential of lithium-ion battery particles. Particle and Fibre Toxicology, 16, 35.

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Functional Characterization of CX3CR1+ Cells

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Sorted CX₃CR1 cell populations from naïve and tumor bearing mice (purity ≥90%) were co-cultured at the indicated ratios with 10⁶ naïve splenocytes. Plated splenocytes were stimulated with 1 μg anti-CD3 (145-2C11) (BioXCell, West Lebanon, NH USA) and culture supernatants were collected after 72 hours and analyzed for IFN-γ production using murine DuoSet ELISA (R&D Systems, Minneapolis, MN USA). Additionally, sorted populations were plated at 1.5–3×10⁶ cells per well with or without 100 ng/ml LPS stimulation (Sigma Aldrich, St. Louis, MO) and cultured for 72 hours. Supernatants were assessed for IL-10 production per 10⁵ cells using a murine DuoSet ELISA (R&D Systems, Minneapolis, MN USA).

Hart K.M., Usherwood E.J, & Berwin B.L. (2014). CX3CR1 Delineates Temporally and Functionally Distinct Subsets of Myeloid-Derived Suppressor Cells in a Mouse Model of Ovarian Cancer. Immunology and cell biology, 92(6), 499-508.

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Serum AFP and OPN Levels Quantification

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Serum samples were analyzed to determine the levels of alpha fetoprotein (AFP) and osteopontin (OPN) by human alpha-fetoprotein Duo Set Elisa (R&D Systems, DY1369, Minneapolis, MN 55413, USA) and human osteopontin Duo Set Elisa (R&D Systems, DY1433, Minneapolis, MN 55413, USA).

Macek Jílková Z., Seigneurin A., Coppard C., Ouaguia L., Aspord C., Marche P.N., Leroy V, & Decaens T. (2020). Circulating IL-13 Is Associated with De Novo Development of HCC in HCV-Infected Patients Responding to Direct-Acting Antivirals. Cancers, 12(12), 3820.

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Quantification of IFN-lambda Cytokines

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ELISA was carried out using commercial reagents. Levels of hIFN-λ₁ were evaluated by human IL-29/IL-28B (IFN-lambda 1/3) DuoSet ELISA (DY1598B, R&D Systems, USA), according to manufacturer’s instructions. A different commercial kit, Mouse IL-28 A/B (IFN-lambda 2/3) DuoSet ELISA (DY1789B, R&D Systems), was used to assess cross-reactivity.

Shaldzhyan A., Zabrodskaya Y., Yolshin N., Kudling T., Lozhkov A., Plotnikova M., Ramsay E., Taraskin A., Nekrasov P., Grudinin M, & Vasin A. (2021). Clean and folded: Production of active, high quality recombinant human interferon-λ1. Process Biochemistry (Barking, London, England), 111, 32-39.

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