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Sandwich elisa

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The Sandwich ELISA is a type of enzyme-linked immunosorbent assay (ELISA) used to detect and quantify specific proteins in a sample. It involves the immobilization of a capture antibody on a solid surface, followed by the addition of a sample containing the target protein. The target protein is then detected by a second, detection antibody that binds to it. The enzymatic reaction that occurs can be measured to determine the concentration of the target protein in the original sample.

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Lab products found in correlation

Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (2 mentions) Firefly luc one step glow assay, by Thermo Fisher Scientific (2 mentions) Anti cd11c microbeads, by Miltenyi Biotec (2 mentions) Collagenase type 4, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Concanavalin a (cona), by Merck Group (2 mentions) Np20 bsa, by Biosearch Technologies (1 mentions) Anti isotype specific antibodies, by BD (1 mentions) Magellantm, by Tecan (1 mentions) Brefeldin a, by BioLegend (1 mentions) Cd11c beads, by Miltenyi Biotec (1 mentions) Legendplex mouse inflammation panel multi analyte flow assay kit, by BioLegend (1 mentions) Hek blue cell, by InvivoGen (1 mentions) Quanti blue, by InvivoGen (1 mentions) Gen5 plate reader, by Agilent Technologies (1 mentions) Automacs instrument, by Miltenyi Biotec (1 mentions) Histopaque 1083, by Merck Group (1 mentions) Tetramethyl benzidine peroxidase substrate system, by Thermo Fisher Scientific (1 mentions) Maxisorp plate, by Thermo Fisher Scientific (1 mentions) Automacs separator, by Miltenyi Biotec (1 mentions)

30 protocols using sandwich elisa

1

Cytokine Levels in Cell Supernatants

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Levels of IL‐1β, IL‐6, and TNF‐α cytokines in the supernatants were determined by sandwich ELISA (BD Biosciences, CA, USA) according to the manufacturer's instructions.
Kodar K., Harper J.L., McConnell M.J., Timmer M.S, & Stocker B.L. (2017). The Mincle ligand trehalose dibehenate differentially modulates M1‐like and M2‐like macrophage phenotype and function via Syk signaling. Immunity, Inflammation and Disease, 5(4), 503-514.
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2

Sandwich ELISA for Antibody Analysis

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Sandwich ELISA for measuring preimmune sera antibodies using anti-isotype–specific antibodies (BD Pharmingen) to capture IgM, IgG1, IgG2b, or IgG3 were done as described (Zahn et al., 2013 (link)). Antigen-specific antibodies were captured from immunized mice sera by coating ELISA plates with NP20-BSA (Biosearch Technologies) or CGG (100 ng/well; Biosearch Technologies) followed by the detection of IgG1, as described (Zahn et al., 2013 (link)). Sodium thiocyanate NaSCN displacement ELISA to measure antibody affinity/avidity was performed as described (Zahn et al., 2013 (link)), on plates coated as above. Sera were previously titrated by antigen-specific ELISA to choose a working dilution that ensured similar levels of antigen-specific antibodies across samples. Relative affinity values were calculated as described (Zahn et al., 2013 (link)).
Litzler L.C., Zahn A., Dionne K.L., Sprumont A., Ferreira S.R., Slattery M.R., Methot S.P., Patenaude A.M., Hébert S., Kabir N., Subramani P.G., Jung S., Richard S., Kleinman C.L, & Di Noia J.M. (2023). Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice. The Journal of Experimental Medicine, 220(9), e20220381.
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3

Quantifying Inflammatory Biomarkers in Plasma and SF

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Plasma and SF C5a/C5a desArg were measured by a sandwich ELISA from BD Bioscience. In brief, microtiter plates were precoated with monoclonal antibody specific for human C5a/C5a desArg. A standard was supplied with the kit containing lyophilized human serum containing a specific amount of C5a desArg. A calibrator curve was prepared by spiking C5a desArg standard into standard diluent (animal serum), ranging from 0.078 ng/ml to 5 ng/ml. In addition, three quality control samples (QCs) were prepared covering Low (0.25 ng/ml), Medium (2 ng/ml) and High (4 ng/ml) level of the standard curve. Any C5a/C5a desArg in the samples was bound to the plate, and was detected by a mixture of biotinylated anti-human C5a antibody and streptavidin-HRP followed by colorimetric detection using Tetramethylbenzidine. The plate was read at 450 nm and reference at 620 nm on a Tecan Sunrise reader controlled by MagellanTM, Tecan Trading AG, Switzerland. The instrument response was exported to WatsonLIMSTM software, ThermoFishes Scientific, where plasma concentrations were quantified.
SFs from RA patients were sent to Myriad RBM for analysis on their Human DiscoveryMAP® 175+ v1.0, a 45-biomarker Multi-Analyte Profile (MAP) designed to discern inflammatory biomarker patterns.
Hornum L., Hansen A.J., Tornehave D., Fjording M.S., Colmenero P., Wätjen I.F., Søe Nielsen N.H., Bliddal H, & Bartels E.M. (2017). C5a and C5aR are elevated in joints of rheumatoid and psoriatic arthritis patients, and C5aR blockade attenuates leukocyte migration to synovial fluid. PLoS ONE, 12(12), e0189017.
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4

Cytokine Release Modulation in Immune Cells

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After pre-stimulation with CS extract, HaCaT cells were treated with TNF‑α and IFN‑γ. Splenocytes were pre-treated with or without CS and then stimulated with 1 μg/mL concanavalin A (Merck, Kenilworth, NJ, USA) for 24 h and 48 h. Cell supernatants were collected and the concentrations of TARC, IL-6, IL‑8, MCP‑1, IL‑4, IL‑5, IL‑13, and eotaxin were measured in the supernatant by sandwich ELISA (BD Biosciences, San Jose, CA, USA and R&D Systems).
Cha K.J., Song C.S., Lee J.S., Kashif A., Hong M.H., Kim G, & Kim I.S. (2019). Chaenomeles sinensis Koehne extract suppresses the development of atopic dermatitis-like lesions by regulating cytokine and filaggrin expression in NC/Nga mice. International Journal of Medical Sciences, 16(12), 1604-1613.
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5

Macrophage Infection Assay for Tularemia

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Murine macrophages or PMA-differentiated THP-1 cells were plated to a density of 1 × 105 cells per well in flat bottomed 96 well plates. Cells were infected (MOI 40) for 2 h with F. tularensis live vaccine strain (LVS). Following infection, cells were washed twice with PBS pH 7.2, cultured in fresh culture media containing 50 µg/mL gentamycin and incubated for an additional 16–24 h. For compound testing, cells were incubated with 1 mM to 1 nM final concentration of test compound for 2 h pre-infection or for 24 h post-infection in RPMI media containing 50 µg/mL gentamycin. The following day conditioned media was collected and stored at −20 °C for cytokine analysis. Pro-inflammatory cytokines (IL-1β and IL-6) were independently measured by sandwich ELISA following manufacturer’s protocol (BD Bioscience, San Jose, CA). For each experiment, 50 µL of undiluted conditioned media was assayed.
Kulkarni A.A., Sajith A.M., Duarte T.T., Tena A., Spencer C.T, & Bowen J.P. (2019). Design, synthesis, and screening of sulfonylurea-derived NLRP3 inflammasome inhibitors. Medicinal Chemistry Research, 29(1), 126-135.
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6

Evaluating CAR T cell Cytotoxicity

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The luciferase-based cytotoxicity assay was carried out as previously described [38 (link)]. On day 7 post-transduction, effector CAR T cells were co-cultured with target MCF-7 (Her2+) tumour cells at a 10:1 ratio of effector to target (E: T) cells in triplicate using Firefly Luc One-Step Glow assay (Thermo Fisher #16197). To evaluate the role of TNF-α in CAR T cells killing breast cancer cell line, 5.0 × 106 of each CAR T cell constructs were treated with 100 μg/mL infliximab (IFX) and incubated for 24 h at 37 °C, 5% CO2 prior to cytotoxicity assay. For IL-2 and IFN-γ cytokine release assay, CAR T cells were stimulated with Her2+ target cells at 2:1 ratio. The concentration of cytokines secreted in cell supernatant was measured 24 h post-incubation using sandwich ELISA according to manufacturer’s protocol (BD Biosciences, San Jose, CA, USA). Plates were read on a Varioskan Lux multimode microplate reader (Thermo Fisher, USA). To measure intracellular cytokines, perforin and granzyme, CAR T cells were co-incubated with target cells at 2:1 ratio for 6 h and brefeldin A for 4 h (Biolegend, San Diego, CA, USA, #420601) prior to intracellular labelling.
Tan G.M., Poudel A., Ali Hosseini Rad S.M, & McLellan A.D. (2022). Anti-Apoptotic c-FLIP Reduces the Anti-Tumour Activity of Chimeric Antigen Receptor T Cells. Cancers, 14(19), 4854.
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7

Cytokine Profile Determination in Mice

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Determination of serum levels of interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP1) of mice treated with ASO-1560S or ASO-C or left untreated was carried out by sandwich ELISA (BD Biosciences, San Diego, CA), according to manufacturer's directions.
Lobos-González L., Silva V., Araya M., Restovic F., Echenique J., Oliveira-Cruz L., Fitzpatrick C., Briones M., Villegas J., Villota C., Vidaurre S., Borgna V., Socias M., Valenzuela S., Lopez C., Socias T., Varas M., Díaz J., Burzio L.O, & Burzio V.A. (2016). Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors. Oncotarget, 7(36), 58331-58350.
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8

Measuring Cytokine Responses to Mycobacterial Infection

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Per the methods of Chackerian et al (43 (link)), ½ of each spleen from Mtb-infected VitDHI and VitDLO mice were collected at the indicated time points, pushed through a 70μm nylon screen and treated with sterile RBC lysis buffer. 2×106 cells per spleen in 2mL of complete RPMI media (RPMI + 10% FBS) were then stimulated with 1μg/mL concanavalin A (ConA) and live Mtb (H37Rv) at MOI=0, 0.5 or 1 for 48 hours. Supernatants were then collected, filtered through a 0.22μm filter and stored at −80°C until being assayed for murine IFNγ and IL12/23p40 by sandwich ELISA (BD Biosciences).
Reeme A.E, & Robinson R.T. (2016). Dietary Vitamin D3 suppresses pulmonary immunopathology associated with late stage tuberculosis in C3HeB/FeJ mice. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1293-1304.
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9

Cytotoxicity and Cytokine Profiling of CAR T Cells

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Luciferase-based cytotoxicity assay was carried out for Her2 and CD19 CAR T cells as previously described [22 ] at a 10:1 ratio of effector to target cells of using Firefly Luc One-Step Glow assay (Thermo Fisher #16197). For analysis of cytokine release, CAR T cells were added to target cells in a 2:1 ratio. IL-2 and IFN-γ concentration secreted in cell supernatant were measured using sandwich ELISA according to manufacturer’s protocol (BD Biosciences, USA). Plates were read on a Varioskan Lux multimode microplate reader (Thermo Fisher, USA).
Rad S. M. A.H., Poudel A., Tan G.M, & McLellan A.D. (2020). Promoter choice: Who should drive the CAR in T cells?. PLoS ONE, 15(7), e0232915.
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10

T Cell Activation Assay Protocol

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1.5 to 2.5×104 T cells were left rested or stimulated with either anti-CD3ε+anti-CD28 antibodies, T cell depleted OVA peptide 323-339 (OVA323-339)-loaded splenocytes at a 1:5 T cell:splenocyte ratio, or CD11c+ purified dendritic cells (using CD11c-beads; Miltenyi Biotech) loaded with OVA323-339 or melanoma tumor lysates at a 1:3 dendritic cell:T cell ratio. Culture supernatants were typically harvested 24 hours after stimulation, and IL-2 or IFNγ levels were measured by a sandwich ELISA (BD Biosciences).
Bandyopadhyay S., Quinn T.J., Scandiuzzi L., Basu I., Partanen A., Tomé W.A., Macian F, & Guha C. (2016). Low intensity focused ultrasound induces reversal of tumor-induced T cell tolerance and prevents immune escape. Journal of immunology (Baltimore, Md. : 1950), 196(4), 1964-1976.
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