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Sandwich elisa kit

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Sandwich ELISA kits are a type of enzyme-linked immunosorbent assay (ELISA) used for the detection and quantification of target analytes in a sample. The kit utilizes a capture antibody coated on a solid support, which binds the target analyte. A detection antibody then binds to the captured analyte, forming a 'sandwich' structure. The enzyme-linked detection antibody catalyzes a colorimetric or fluorometric reaction, allowing for the measurement of the target analyte concentration.

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Lab products found in correlation

Tgf β1, by Cell Signaling Technology (2 mentions) Tgfβ1, by RayBiotech (2 mentions) C series 1000, by RayBiotech (2 mentions) Multiskan go, by Thermo Fisher Scientific (2 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions) Cell culture insert, by Merck Group (1 mentions) Tgf β1, by Merck Group (1 mentions) Microplate reader, by Tecan (1 mentions) Protease inhibitor, by Merck Group (1 mentions)

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ELISAs (7 citations) Sandwich ELISA (6 citations) Antibody sandwich ELISA kit (2 citations) VEGF-A sandwich ELISA kit (human and rat VEGF-A kits, (2 citations)

20 protocols using sandwich elisa kit

1

Cytokine Quantification in Serum and Culture

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TNFα and IL-1β concentrations in the serum samples and culture supernatants were measured using sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) as reported (17 (link)). Soluble TNFR1 (sTNFR1) concentrations in the culture supernatants and blood serum were measured using sandwich ELISA kits (RayBio, Norcross, GA, USA). All the kits were used in accordance with the manufacturer’s protocol. The optical density of each well was measured at 450 nm using a microplate reader (Varioskan Flash, Thermo Fisher Scientific), and the concentrations of each sample were calculated based on standard curves.
Akagi T., Hiramatsu-Asano S., Ikeda K., Hirano H., Tsuji S., Yahagi A., Iseki M., Matsuyama M., Mak T.W., Nakano K., Ishihara K., Morita Y, & Mukai T. (2022). TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1. Frontiers in Immunology, 13, 926175.
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2

Hydrogel-Mediated TGFβ1 Release Kinetics

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Hydrogel macromers of AcHyA (13.3 mg/mL), AcHyA-RGD (20 mg/mL), and heparin-SH (0.013 mg/mL) were dissolved at various ratios (Supplementary table S1) in 0.3 mL of triethanolamine-buffer (TEOA; 0.3 M, pH 8) and incubated for 15 minutes at 37 °C. Then, TGFβ1 (350 ng, Cell Signaling Technology, Danvers, MA) was mixed in the solution of HyA derivatives and incubated for another 15 min at 37°C. To determine the release kinetics, TGFβ1 containing HyA hydrogels were transferred to cell culture inserts (Millipore Corporation, Billerica, MN) and TGFβ1 was allowed to release into 400 μL of cell culture media per well. At predetermined time points over the course of 3 weeks, the supernatant was withdrawn and fresh media was replenished, and the mass of TGFβ1 in each supernatant was determined with sandwich ELISA kits (RayBiotech, Inc, Norcross GA) (Supplementary Fig 5a, b). Retention of TGFβ1 was calculated by the subtraction of released TGFβ1 from the calculated initial loading amount of TGFβ1 (Supplementary Fig 5a). Similarly, retention kinetic of bovine serum albumin (BSA) from HyA hydrogels (3wt% with 100% crosslinked without heparin) was measured (Supplementary Fig 5b).
Jha A.K., Tharp K.M., Ye J., Santiago-Ortiz J.L., Jackson W.M., Stahl A., Schaffer D.V., Yeghiazarians Y, & Healy K.E. (2015). Enhanced Survival and Engraftment of Transplanted Stem Cells using Growth Factor Sequestering Hydrogels. Biomaterials, 47, 1-12.
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3

CXCL9 and CXCL10 Measurement by ELISA

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Cell supernatants were tested for CXCL9 and CXCL10 content using DuoSet sandwich ELISA kits from RnD Systems. Plasma and serum levels of CXCL9 and CXCL10 were determined by sandwich ELISA kits validated for plasma/serum analyses from RayBiotech, Inc.
Edvardsen K., Bjånesøy T., Hellesen A., Breivik L., Bakke M., Husebye E.S, & Bratland E. (2015). Peripheral Blood Cells from Patients with Autoimmune Addison's Disease Poorly Respond to Interferons In Vitro, Despite Elevated Serum Levels of Interferon-Inducible Chemokines. Journal of Interferon & Cytokine Research, 35(10), 759-770.
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4

Multiplex ELISA and SARS-CoV-2 Antibody Assay

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Multiplex ELISA for 71 cytokines and chemokines was performed on the −80°C stored serum samples, from each time point, using a Multiplexing LASER Bead Assay (Human Cytokine Array/Chemokine Array 71-Plex Panel, HD71, Eve Technologies, Canada) while CCL28, RAGE, SP-D, IL-22BP were determined by Sandwich ELISA kits, as specified by the manufacturer (RayBiotech, GA).
SARS-CoV-2 specific antibody responses were evaluated through quantitative tests for IgM, IgG, and IgA against spike protein or its receptor binding domain, as previously described (18 (link)).
Trombetta A.C., Farias G.B., Gomes A.M., Godinho-Santos A., Rosmaninho P., Conceição C.M., Laia J., Santos D.F., Almeida A.R., Mota C., Gomes A., Serrano M., Veldhoen M., Sousa A.E, & Fernandes S.M. (2021). Severe COVID-19 Recovery Is Associated with Timely Acquisition of a Myeloid Cell Immune-Regulatory Phenotype. Frontiers in Immunology, 12, 691725.
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5

Quantification of EV Biomarkers via ELISA

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GH, PCT, PAI1, PlGF and TIMP1 concentrations were determined by commercially available sandwich ELISA kits (RayBiotech) according to the manufacturer’s instructions. For each biomarker 200 μL of the lysed EVs (as described above) or 30 μL of plasma diluted to 200 μL with dilution buffer were incubated with the respective immobilised primary antibody at 4°C overnight. After three washes, the horseradish peroxidase (HRP)-conjugated secondary antibodies were added and incubated for 3 hours at room temperature. After three washes, tetramethylbenzidine (TMD) solution was added and incubated for 45 minutes. The reaction was stopped by the addition of the acidic “stop” solution provided by the manufacturer and absorbance of the resulting yellow product was measured at λ450 nm, using a Tecan microplate reader. Concentrations were determined from a standard curve generated by the standards provided in the kit.
Tan K.H., Tan S.S., Ng M.J., Tey W.S., Sim W.K., Allen J.C, & Lim S.K. (2017). Extracellular vesicles yield predictive pre-eclampsia biomarkers. Journal of Extracellular Vesicles, 6(1), 1408390.
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6

Hydrogel-Mediated Sustained Release of TGFβ1

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Hydrogel macromers of AcHyA, AcHyA-RGD, and heparin-SH were dissolved at various ratios in 0.3 mL of triethanolamine-buffer (TEOA; 0.3 M, pH 8) for 15 minutes at 37°C. Then, TGFβ1 (Cell Signaling Technology, Inc., Beverly, MA) was mixed in the solution of HyA derivatives and incubated for another 15 min at 4°C. Subsequentely, MMP-13 crosslinker (50 μL TEOA buffer) was added to form TGFβ1 loaded hydrogel, then TGFβ1 was allowed to release into 400 μL of cell culture media. At predetermined time points over the course of 3 weeks, the supernatant was withdrawn and fresh media was replenished. The mass of TGFβ1 in each supernatant was determined with sandwich ELISA kits (RayBiotech, Inc, Norcross GA). Retention of TGFβ1 was calculated by subtraction of released TGFβ1 from the calculated initial loading amount of TGFβ1.
Jha A.K., Mathur A., Svedlund F.L., Ye J., Yeghiazarians Y, & Healy K.E. (2015). Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate. Journal of controlled release : official journal of the Controlled Release Society, 209, 308-316.
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7

Quantifying Retinal GSK3β Levels

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The retinal levels of glycogen synthase kinase-3β (GSK3β) were measured using sandwich ELISA kits (RayBiotech, Peachtree Corners, GA, USA) on day 7 after ONC, according to the manufacturer’s protocols. After euthanizing the animals, retinas were isolated. After homogenizing the retina using T-PER in the presence of protease inhibitor (Merck, Darmstadt, Germany), the homogenized solution was centrifuged at 10,000× g for 10 min at 4 °C and the supernatant was collected. Each well in the 96 well-microplate was precoated with purified GSK3β antibodies. Every standard or sample solution was added into 2 wells to provide duplicate measurements in each sample. Samples were incubated overnight at 4 °C to form the GSK3β antibody-GSK3β complex. After washing, HRP-labeled GSK3β antibody was added into each well. Subsequently, TMB substrate was added to develop a blue color and the color intensity proportionally reflected the amount of GSK3β in each well based on the catalysis of the HRP enzyme activity. Finally, the developed color was converted into yellow using acid. Then, the absorbance (OD) at 450 nm was determined by a micro-plate reader (SH-100 Lab, Corona, Ibaraki, Japan). The GSK3β level was obtained by a calibration curve and standardized by protein concentration.
Fukiyama Y., Hirokawa T., Takai S., Kida T, & Oku H. (2023). Involvement of Glycogen Synthase Kinase 3β (GSK3β) in Formation of Phosphorylated Tau and Death of Retinal Ganglion Cells of Rats Caused by Optic Nerve Crush. Current Issues in Molecular Biology, 45(9), 6941-6957.
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8

CTSL and CTSB Levels Quantification

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Levels of serum CTSL and CTSB were assayed using a commercially available sandwich ELISA kits (RayBiotech, Norcross, GA and Bosterbio, Pleasanton, CA, U.S.A.) as per the manufacturer's instructions as described in detail elsewhere (18 (link)).
Mehra S., Panwar R., Thakur B., Yadav R., Kumar M., Singh R., Dash N.R., Sahni P, & Chauhan S.S. (2019). Expression and Clinical Implications of Cysteine Cathepsins in Gallbladder Carcinoma. Frontiers in Oncology, 9, 1239.
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9

Quantifying Cell-Secreted MMPs and VEGF

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Cell/gel constructs were cultured in 400 μL cell culture media. At predetermined time points over the course of 3 weeks, the surrounding culture media and gels were collected and digested in hyaluronidase (3000 unit/mL). Subsequently, supernatants were collected after centrifugation (3000 rpm, 5 min) of the degraded hydrogels. The mass of MMPs and VEGF165 secreted by the entrained cells in collected supernatant was determined using sandwich ELISA kits (RayBiotech, Inc., Norcross GA).
Jha A.K., Tharp K.M., Browne S., Ye J., Stahl A., Yeghiazarians Y, & Healy K.E. (2016). Matrix Metalloproteinase-13 Mediated Degradation of Hyaluronic Acid-based Matrices Orchestrates Stem Cell Engraftment through Vascular Integration. Biomaterials, 89, 136-147.
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10

Cytokine and Chemokine Profiling

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Cytokine/chemokine contents in culture supernatants were measured using sandwich ELISA kits (Ray Biotech; Peachtree Corners, GA, USA) according to the manufacturer’s protocol. Cytokines/chemokines in mouse plasma were measured using a MILLIPLEX® Human Cytokine/Chemokine Magnetic Bead Panel – Premixed 41 Plex – Immunology Multiplex Assay (HCYTMAG60PMX41BK; Millipore Sigma) and a MILLIPLEX® Mouse Cytokine/Chemokine Magnetic Bead Panel – Premixed 32 Plex – Immunology Multiplex Assay (MCYTMAG-70K-PX32; Millipore Sigma).
Yamakawa T., Zhang G., Najjar L.B., Li C, & Itakura K. (2023). The uncharacterized transcript KIAA0930 confers a cachexic phenotype on cancer cells. Oncotarget, 14, 723-737.
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