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Quantitative sandwich enzyme immunoassay

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The Quantitative sandwich enzyme immunoassay is a laboratory equipment used for the quantitative detection and measurement of specific proteins or analytes in a sample. It utilizes the principles of sandwich ELISA, where the target analyte is captured between two antibodies and detected through an enzymatic reaction. This equipment provides a standardized and reliable method for quantifying the concentration of the target analyte in the sample.

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Lab products found in correlation

Mpo kit, by Miltenyi Biotec (2 mentions) Bio plex manager 6, by Bio-Rad (1 mentions) 24 well tissue culture plate, by BD (1 mentions) Turbidimetric inhibition immunoassay, by Roche (1 mentions) Particle enhanced turbidimetric assay, by Roche (1 mentions) Paramagnetic particle chemiluminescent immunoassay, by Beckman Coulter (1 mentions) Hscrp, by Roche (1 mentions) Luminex x 200 instrument, by R&D Systems (1 mentions) P53 do 1, by Novus Biologicals (1 mentions) Hif 1α, by Novus Biologicals (1 mentions) Vista 1500, by Siemens (1 mentions) Immulite 2000, by Siemens (1 mentions)

14 protocols using quantitative sandwich enzyme immunoassay

1

Diurnal Cytokine Profiling in Fasted Patients

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We collected the blood samples from patients in the morning (between 07.30 and 10.00 a.m.) after 8 hours of fasting due to diurnal variability of CTx serum on the same day they completed the self-report questionnaire. Serum specimens were stored frozen (-200C) until the time of analysis. IL-6, IL-17, and TNF-α were analyzed by quantitative sandwich enzyme immunoassay (R&D systems, UK). Each cytokine assay was performed on one 96-well plate, and the optical density of each was measured using a microplate reader set to 450 nm. The measuring range for each assay was IL-6, 3.1 -300 pg/mL; IL-17, 31.2 – 2,000 pg/mL; and TNF-α, 15.6 – 1,000 pg/mL.
Parlindungan F., Hidayat R., Ariane A, & Shatri H. (2023). Association between Proinflammatory Cytokines and Anxiety and Depression Symptoms in Rheumatoid Arthritis Patients: A Cross-sectional Study. Clinical Practice and Epidemiology in Mental Health : CP & EMH, 19, e174501792304261.
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2

Measuring Endogenous Estradiol and Inflammation

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Serum specimens were used to measure circulating endogenous levels of unconjugated estradiol in addition to 69 inflammation biomarkers spanning multiple aspects of inflammation [29 ] (Supplemental Table 1). Unconjugated estradiol was measured via gas chromatography tandem mass spectrometry [26 (link)]. In a single laboratory, 68 inflammation markers were measured using five Luminex bead-based commercial assay panels (Millipore, Billerica, MA). The 69th marker, transforming growth factor beta-1 (TGF-beta-1), was measured using a quantitative sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN). Marker concentrations were calculated using a four- or five-parameter logistic curve (Bioplex Manager 6.1, BioRad, Hercules, CA). The high-sensitivity panel used the same manufacturer lot, but the remaining panels were purchased across two lots. We evaluated assay performance by including a blinded duplicate sample from a quality control pool in each batch; information on the quality control is previously published [26 (link)]. For the parent study, the estradiol assay coefficient of variation (CV) was 17.1% with an intraclass correlation coefficient (ICC) of 0.97. For the multiplex inflammatory assay, in the parent study, the CVs were on average 9.1% (range 2.7–32.2), with an ICC average of 0.89 (range 0.67–0.99). The study sample CVs are detailed in Supplementary Table 1.
Eldridge R.C., Wentzensen N., Pfeiffer R.M., Brinton L.A., Hartge P., Guillemette C., Kemp T.J., Pinto L.A, & Trabert B. (2020). Endogenous estradiol and inflammation biomarkers: potential interacting mechanisms of obesity-related disease. Cancer causes & control : CCC, 31(4), 309-320.
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3

Quantitative Spinal Cord Cytokine Assay

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Spinal cord was harvested and weighed sections were homogenized in homogenization buffer. Samples were cold centrifuged and supernatant was used for the examination of IL-1β and TNF-α concentration levels using the quantitative sandwich enzyme immunoassay according to the manufacturer's instructions (R&D Systems, MN, USA). The levels of IL-1β and TNF-α were determined by comparing samples to the standard curve generated from the respective kits at 450 nm and were expressed as pg per mg tissue (spinal cord).
Sun J.J., Tang L., Zhao X.P., Xu J.M., Xiao Y, & Li H. (2019). Infiltration of Blood-Derived Macrophages Contributes to the Development of Diabetic Neuropathy. Journal of Immunology Research, 2019, 7597382.
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4

Cytokine Production in HTLV-1 Infected Cells

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MT-2 and HTLV-1 infected CD4+ cells were harvested, counted, suspended in CM and seeded into 24-well tissue culture plates (Falcon) in 1 ml/well at a concentration of 1x105 cell/ ml. The plates were incubated at 37 °C in a 5 % CO2 humidified atmosphere for 30 min, 1, 3, 6, 12, 24, 48 or 72 h. Each culture was set in triplicate. At each time, cells were analyzed for proinflammatory cytokine production. Culture supernatants of MT-2 and HTLV-1 infected CD4 + cells were collected as control for cytokine detection.
The concentrations of IL-1α, IL-6, and TNF-α were measured in undiluted cell culture supernatants in triplicate by multiplex enzyme-linked immunosorbent assay based on SearchLight™ technology (Thermo Fisher Scientific Pierce Searchlight Products, Woburn, MA). The amount of signal produced is proportional to the amount of each protein in the sample detected with a cooled CCD camera. The assay [24 (link)] is capable of detecting target proteins at pg/ml concentrations.
To detect IL-17 production, a quantitative sandwich enzyme immunoassay (R&D Systems). technique specific for natural and recombinant human protein was used. According to the manufacturer, the detection limit of the assays was approximately 15.0 pg/ml.
Fuggetta M.P., Bordignon V., Cottarelli A., Macchi B., Frezza C., Cordiali-Fei P., Ensoli F., Ciafrè S., Marino-Merlo F., Mastino A, & Ravagnan G. (2016). Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol. Journal of Experimental & Clinical Cancer Research : CR, 35, 118.
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5

Lung Tissue Protein Extraction

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The right middle lung lobes were placed in 1 mL of homogenization buffer (4°C). Samples were homogenized and incubated at 4°C for 1 h. The final homogenate was centrifuged at 10,000 rpm for 15 min. Tissue supernatants were used for the determination of MPO and NE activity. MPO activity was measured using an MPO kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. NE activity was determined using a quantitative sandwich enzyme immunoassay (R&D Systems, USA) following the manufacturer's instructions.
Gan L., Zhong J., Zhang R., Sun T., Li Q., Chen X, & Zhang J. (2015). The Immediate Intramedullary Nailing Surgery Increased the Mitochondrial DNA Release That Aggravated Systemic Inflammatory Response and Lung Injury Induced by Elderly Hip Fracture. Mediators of Inflammation, 2015, 587378.
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6

Prolactin Measurement by Enzyme Immunoassay

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Prolactin was measured by quantitative sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN) at the section of Analytical Chemistry and Neurochemistry, Department of Chemistry, Uppsala University, in one batch. The limit of detection was 0.6 ng/mL. The relative SD from blinded replicate samples was less than 6%.
Gabrielson M., Ubhayasekera K., Ek B., Andersson Franko M., Eriksson M., Czene K., Bergquist J, & Hall P. (2018). Inclusion of Plasma Prolactin Levels in Current Risk Prediction Models of Premenopausal and Postmenopausal Breast Cancer. JNCI Cancer Spectrum, 2(4), pky055.
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7

Metabolic and Inflammatory Biomarker Assessment

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We measured biomarkers of metabolic functioning [hemoglobin A1C (HbA1C) and adiponectnin], and inflammation [high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a)]. HbA1C was measured by turbidimetric inhibition immunoassay (Roche Diagnostics, Indianapolis, IN). Adiponectin was measured by enzyme-linked immunosorbent assay (ELISA) (ALPCO Diagnostics Inc, Salem, NH). hs-CRP was measured using particle enhanced turbidimetric assay (Roche Diagnostics, Indianapolis, IN). IL-6 was measured by paramagnetic particle, chemiluminescent immunoassay (Beckman Coulter, Fullerton, CA). TNF-a was measured using quantitative sandwich enzyme immunoassay (R&D Systems Inc., Minneapolis, MN).
Chacko S.A., Yeh G.Y., Davis R.B, & Wee C.C. (2016). A mindfulness-based intervention to control weight after bariatric surgery: Preliminary results from a randomized controlled pilot trial. Complementary therapies in medicine, 28, 13-21.
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8

Multiplex Cytokine and Biomarker Profiling

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Serum levels of 35 cytokines, including C-C motif ligand (CCL)2, CCL4, CCL11, CCL20, CCL25, CCL26, CX3CL1, CXCL8, CXCL11, ADAMTS13, adiponectin, α-2-macroglobulin, matrix metalloproteinase (MMP)3, insulin-like growth factor binding protein-1, TNF-α, IL-1ß, IL-2, IL-4, IL-6, IL-7, IL-10, IL-11, IL-12 p70, IL-13, IL-17, IL-18, IL-23, IL-27, IL-28A, IL-28B, IL-31, IL-33, IL-34, and IL-36ß, at baseline and week 2 in discovery cohort were measured using a Luminex cytokine assay kit (LXSAHM-35) and a Luminex X-200 instrument according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).
Enzyme-linked immunosorbent assay (ELISA) was used to measure levels of the molecules selected after the analysis of Luminex results obtained in the discovery cohort. In cohort 1 and 2, serum MMP3 levels were measured using a quantitative sandwich enzyme immunoassay (R&D Systems) at baseline and week 2. The serum CCL2 concentration was measured at week 2 using an ELISA kit (Abcam, Cambridge, UK, Ab179886). Serum IL-7 levels were measured using an ELISA kit (R&D Systems). Other indicators, such as CRP, ESR, albumin and so on, were tested with standard institutional protocols by the laboratory physicians at the First Affiliated Hospital of Sun Yat-sen University.
Li L., Chen R., Zhang Y., Zhou G., Chen B., Zeng Z., Chen M, & Zhang S. (2021). A Novel Model Based on Serum Biomarkers to Predict Primary Non-Response to Infliximab in Crohn’s Disease. Frontiers in Immunology, 12, 646673.
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9

Western Blot Analysis of Hypoxia Signaling

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Western blots were performed as described earlier [19 (link)]. Membranes were probed with the primary antibodies: p21 ((C-19)-G), p53 (DO-1), p-p53, HIF-1α (Novus Biologicals, Littleton, USA), gamma-H2AX, TWIST and GAPDH followed by horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG-HRP (Santa-Cruz, California, USA). All blots have been generated from triplicate experiments after 6 hours of exposure to DCQ or hypoxia. The concentration of VEGF in the culture supernatant was measured using a quantitative sandwich enzyme immunoassay (R&D Systems Inc., Minneapolis, USA) according to the manufacturer’s instructions. VEGF concentrations were calculated by comparison with a standard curve generated using a 4-parameter logistic curve-fit and on-board software. VEGF concentrations were normalized to amount (pg) per 1 × 106 living cells/10 ml supernatant.
Ghattass K., El-Sitt S., Zibara K., Rayes S., Haddadin M.J., El-Sabban M, & Gali-Muhtasib H. (2014). The quinoxaline di-N-oxide DCQ blocks breast cancer metastasis in vitro and in vivo by targeting the hypoxia inducible factor-1 pathway. Molecular Cancer, 13, 12.
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10

Serum Biomarker Profiling: IL-6, CRP, TNF-aR1

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Non-fasting blood (serum) was drawn at baseline. Aliquots were stored, using customary secure procedures, at −70° C at both field centers. For this analysis, the following soluble protein biomarkers were measured: IL-6, CRP, and TNF-aR1. IL-6 was measured using a solid-phase immunometric assay on the IMMULITE 2000 (Siemens Healthineers). Assay detection was conducted via an enzyme-labeled, sequential assay in which a substrate was converted to the chemiluminescent product. The total imprecision of this method was acceptable as indicated by a 4-6% between-run coefficient of variation. High-sensitivity CRP (hsCRP) was measured using a Vista 1500 (Siemens Healthineers) by immunonephelometry in which polystyrene particles coated with monoclonal antibodies against human CRP aggregate with this protein in samples. The between-run imprecision (coefficient of variation) was typically 4 to 6%. TNF-aR1 was measured using a quantitative sandwich enzyme immunoassay (R&D Systems, Inc) where assay detection was colorimetric. Total imprecision of the runs was acceptable as indicated by a 3-4% between-run coefficient of variation.
Wanigatunga A.A., Chiu V., Cai Y., Urbanek J.K., Mitchell C.M., Miller ER I.I.I., Christenson R.H., Rebuck H., Michos E.D., Juraschek S.P., Walston J., Xue Q.L., Bandeen-Roche K., Appel L.J, & Schrack J.A. (2022). Patterns of Daily Physical Movement, Chronic Inflammation, and Frailty Incidence. Medicine and science in sports and exercise, 55(2), 281-288.
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