High sensitivity elisa kit

Manufactured by R&D Systems

Sourced in United States, United Kingdom, Australia

High-sensitivity ELISA kits are laboratory equipment designed to measure the concentration of specific target analytes in a sample with high precision and accuracy. These kits utilize the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the presence of target molecules, such as proteins, hormones, or other biomolecules, in a variety of sample types.

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46 protocols using high sensitivity elisa kit

Inflammatory Markers Measurement Protocol

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The inflammation composite was a mean of the standardized scores of four inflammatory markers, interleukin 6 (IL-6), C-reactive protein (CRP), interleukin 1 receptor antagonist (IL-1RA), and macrophage migration inhibitory factor (MIF). IL-6 was measured using a high-sensitivity ELISA kit (R&D Systems, Minneapolis, MN) (intra-assay CV<10%; detection threshold=.04 pg/ml). CRP was measured using a high-sensitivity, chemiluminescent technique on an IMMULITE 2000 (Diagnostic Products Corporation, Los Angeles, CA) (inter-assay CV=2.2%; detection threshold=.20 mg/L). IL1RA was measured using a high-sensitivity ELISA kit (R&D systems (R&D Systems, Minneapolis, MN) (intra-assay CV<10%; detection threshold = 18.3 pg/mL). MIF was measured using using a high-sensitivity ELISA kit (R&D systems (R&D Systems, Minneapolis, MN) (intra-assay CV<10%; detection threshold = 31.2 pg/mL).

Levine C.S., Basu D, & Chen E. (2016). Just World Beliefs are Associated with Lower Levels of Metabolic Risk and Inflammation and Better Sleep After an Unfair Event. Journal of personality, 85(2), 232-243.

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Biomarkers of Chronic Inflammation

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At baseline, fasting blood samples were processed at the research clinic for immediate measurement of C-reactive protein (CRP), IL-6, TNF-α and fibrinogen. CRP was assayed using a high-sensitivity immunonephelometric assay (Dade Behring, UK) and TNF-α and IL-6 antigen levels were determined using high-sensitivity ELISA kits (R&D Systems, UK), performed in the University Department of Medicine, Glasgow Royal Infirmary. Participants with CRP level above 10 mg/l were removed from analyses, as this is indicative of acute inflammation likely as a result of a temporary infection and not chronic low-level inflammation.

Sluiman A.J., McLachlan S., Forster R.B., Strachan M.W., Deary I.J, & Price J.F. (2021). Higher baseline inflammatory marker levels predict greater cognitive decline in older people with type 2 diabetes: year 10 follow-up of the Edinburgh Type 2 Diabetes Study. Diabetologia, 65(3), 467-476.

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Plasma Biomarker Quantification Protocol

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Plasma was stored at −80°C prior to batch testing. High sensitivity ELISA kits were used to quantitate levels of IL-6 (R&D Systems), D-dimer (Diagnostica-Stago), and soluble CD14 (sCD14, R&D Systems), according to the manufacturers’ instructions. Plasma concentrations of kynurenine and tryptophan were determined by liquid chromatography and mass spectroscopy [32] (link).

Somsouk M., Dunham R.M., Cohen M., Albright R., Abdel-Mohsen M., Liegler T., Lifson J., Piatak M., Gorelick R., Huang Y., Wu Y., Hsue P.Y., Martin J.N., Deeks S.G., McCune J.M, & Hunt P.W. (2014). The Immunologic Effects of Mesalamine in Treated HIV-Infected Individuals with Incomplete CD4+ T Cell Recovery: A Randomized Crossover Trial. PLoS ONE, 9(12), e116306.

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Blood Biomarkers and Insulin Resistance

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Blood was transferred into tubes containing 0.3 mol·L⁻¹ EDTA (10 μL·ml⁻¹ blood) and immediately centrifuged at 4°C for 10 min at 23,000 g. The plasma was stored at –80°C until analysis. Analysis of plasma insulin and glucose has been described previously (Ara et al., 2011 (link)). The Quicki index was calculated as previously described (Katz et al., 2000 (link)). Blood HbA1c was analyzed on a DCA Vantage Analyzer (Siemens Healthcare, NY, USA). Plasma IL-6 and plasma TNF-α were measured with high-sensitivity ELISA kits from R&D Systems (Minneapolis, MN, USA). The plasma IL-18 was analyzed with HS ELISA (R&D Systems) and CRP using a conventional high-sensitivity assay on a Cobas Hitachi (Roche, Indianapolis, USA). The plasma adiponectin concentration was determined with a human radioassay kit (EMD Millipore. Billerica, MA, USA). The plasma leptin concentration was measured by a high-sensitivity human ELISA kit (Human leptin Immunoassay, R&D systems).

Sahl R.E., Andersen P.R., Gronbaek K., Morville T.H., Rosenkilde M., Rasmusen H.K., Poulsen S.S., Prats C., Dela F, & Helge J.W. (2017). Repeated Excessive Exercise Attenuates the Anti-Inflammatory Effects of Exercise in Older Men. Frontiers in Physiology, 8, 407.

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Serum Biomarker Quantification Protocol

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Serum concentrations of TNF-α and IL-6 were measured using high-sensitivity ELISA kits (R&D Systems, Minneapolis, MN) in duplicate (CV <10%). Serum high-sensitivity CRP was measured by latex-enhanced immunonephelometry (Quest Diagnostics; assay CV < 8%).

Flock M.R., Skulas-Ray A.C., Harris W.S., Gaugler T.L., Fleming J.A, & Kris-Etherton P.M. (2014). Effects of supplemental long-chain omega-3 fatty acids and erythrocyte membrane fatty acid content on circulating inflammatory markers in a randomized controlled trial of healthy adults. Prostaglandins, leukotrienes, and essential fatty acids, 91(4), 161-168.

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Quantification of Inflammatory Biomarkers

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The serum or cell supernatant levels of the inflammatory cytokines, including inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α), as well as MMP2 were analyzed using commercially available high sensitivity ELISA kits (R&D Systems Europe, Ltd.). Their concentrations were measured in accordance with their corresponding standard curves. Experiments were independently performed in triplicate.

Luo X., Pan C., Guo X., Gu C., Huang Y., Guo J., Zeng Y., Yue J, & Cui S. (2022). Methylation Mediated Silencing of miR-155 Suppresses the Development of Preeclampsia In Vitro and In Vivo by Targeting FOXO3. Mediators of Inflammation, 2022, 4250621.

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Cytokine Quantification by ELISA

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The IL-6 (cat. no. M6000B), IL-1β (cat. no. RLB00) and TNF-α (cat. no. RTA00) levels in the culture supernatant were analyzed using commercially available high sensitivity ELISA kits (R&D Systems Europe, Ltd.) according to manufacturer's protocol. A microplate reader measured the optical density at 450 nm and the data were used to calculate the concentrations of various cytokines based on the standard curves. The values were determined in three independent experiments.

Liu B., Wei H., Lan M., Jia N., Liu J, & Zhang M. (2020). MicroRNA-21 mediates the protective effects of salidroside against hypoxia/reoxygenation-induced myocardial oxidative stress and inflammatory response. Experimental and Therapeutic Medicine, 19(3), 1655-1664.

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Cytokine Responses to Ultra-Marathon

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Before, immediately after and 90 minutes after completion of the ultra-marathon, blood samples were taken for determination of the plasma concentrations of IL-6, IL-10, IL-18, IL-1β and tumour necrosis factor alpha (TNF-α). The blood samples were collected in tubes containing EDTA. All samples were centrifuged within 15 minutes at 3000 rpm at 4°C, and stored at -70°C until further processed. High-sensitivity ELISA kits provided by R&D Systems (Minneapolis, MN, USA) were used for the pre-race blood samples for IL-6, IL-10 and TNF-α (Quantikine HS ELISA Human IL-6 Immunoassay, Quantikine HS ELISA Human IL-10 Immunoassay and Quantikine HS ELISA Human TNF-α Immunoassay, respectively) because these cytokines exist at very low levels in peripheral blood. Quantitative sandwich ELISA kits were used for the post-race and recovery blood samples for IL-6, IL-10, IL-18, IL-1β and TNF-α (R&D Systems Inc.; Minneapolis, MN, USA). Plasma concentration of IL-18 was determined using a Human IL-18 Instant ELISA kit (eBioscience, Bender MedSystems GmbH, Austria). All samples and provided standards were analysed in duplicate. Haematocrit was measured in duplicate. Whole blood samples (∼9 µl) were transferred to heparinized microcapillary tubes and analysed by an automated system following microcentrifugation. Haematocrit was used to calculate percent changes in plasma volume.

Krzemiński K., Buraczewska M., Miśkiewicz Z., Dąbrowski J., Steczkowska M., Kozacz A, & Ziemba A. (2015). Effect of ultra-endurance exercise on left ventricular performance and plasma cytokines in healthy trained men. Biology of Sport, 33(1), 63-69.

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Comprehensive Cytokine Profiling

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The systemic concentrations of interleukins (IL-1, IL-6, IL-8, IL-10, IL-12, IL-17, and IL-23), G-CSF, and TNFα were measured using commercially available, high-sensitivity ELISA kits (R&D Systems, Minneapolis, MN, USA or BD Bioscience OptEIA ELISA Kits, MD, USA) according to the manufacturer instructions.

Błogowski W., Deskur A., Budkowska M., Sałata D., Madej-Michniewicz A., Dąbkowski K., Dołęgowska B, & Starzyńska T. (2014). Selected Cytokines in Patients with Pancreatic Cancer: A Preliminary Report. PLoS ONE, 9(5), e97613.

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Inflammatory Biomarkers for CHD Risk

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We assessed the extent of low-grade, chronic inflammation using serum levels of C-reactive protein (CRP) and interleukin-6 (IL-6). These are the two most widely used biomarkers of systemic inflammation, and they have been shown to forecast the development of CHD, above and beyond traditional risk factors (Libby, 2012 ; Yeh & Willerson, 2003 (link)). Blood was collected into Serum-Separator Tubes (Becton-Dickinson, Oakville, Ontario) and following the manufacturer’s guidelines, left to clot for 60 min, followed by centrifugation for 10 min at 1000 x g. The serum was harvested and frozen at −80° C until analysis. CRP was measured using a high-sensitivity, chemiluminescent technique on an IMMULITE 2000 (Diagnostic Products Corporation). The CRP assay had an average inter-assay coefficient of variation (CV) of 2.2% and a detection threshold of .20 mg/L. IL-6 was assayed using commercially available high-sensitivity ELISA kits from R&D Systems, which had a detection threshold of 0.04 pg/ml and an average intra-assay CV of 6.19%.

Hostinar C.E., Ross K.M., Chen E, & Miller G.E. (2014). Modeling the Association between Lifecourse Socioeconomic Disadvantage and Systemic Inflammation in Healthy Adults: The Role of Self-Control. Health psychology : official journal of the Division of Health Psychology, American Psychological Association, 34(6), 580-590.

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