Cytokines in NK-cell-conditioned media were quantified using Bead CD8+ T cells Magnetic Bead Panel, Cytokine/Chemokine Magnetic Bead Panel, Cytokine/Chemokine Magnetic Panel III, Th17 Magnetic Bead Panel, or Cytokine/Chemokine Panel II (all human; Merck Millipore, Burlington, MA, USA). Plates were read on Luminex® 200 system, and data were analyzed with Procarta Plex™ Analyst 1.0 (Affymetrix, Waltham, MA, USA). Cytokine levels were normalized to the number of plated cells. The levels of human galectin-9 in the conditioned media of NK-cells were quantified with a sandwich ELISA following the manufacturer’s instructions (RayBiotech, Peachtree Corners, GA, USA). Plates were read on a Spark microwell plate reader (Tecan, Mannedorf, Switzerland).
Sandwich elisa
Manufactured by RayBiotech
Sourced in United States
The Sandwich ELISA is a laboratory equipment used for the quantitative detection and measurement of specific proteins or analytes in a sample. It utilizes the principle of enzyme-linked immunosorbent assay (ELISA) to capture the target analyte between two antibodies, creating a 'sandwich' structure. The Sandwich ELISA provides a sensitive and reliable method for analyzing the presence and concentration of the target analyte in various biological samples.
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6 protocols using sandwich elisa
1
Quantification of Cytokines in NK-Cell Conditioned Media
2
Cytokine and Ig Profiling in Serum and BAL
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The levels of cytokines IL-4, IL-5 and IFNγ, as well as IgG and IgE levels were measured in duplicates in serum and BAL fluid supernatant by sandwich ELISA (RayBiotech Inc, USA), as per manufacturer’s protocol.
3
Quantification of FGF and BMP Proteins
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Levels of FGF 3, FGF10and BMP4 proteins in cell supernatants were determined with sandwich ELISA (RayBiotech, Inc., Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Briefly, for each protein; standard proteins and samples collected from media culture were added to antibody coated plates and incubated overnight at 4 °C. Biotinylated detection antibodies were added to the plates, and incubated at room temperature for 2 h, followed by incubation with peroxidase-labelled streptavidin. Absorbance was measured at 490 nm by ELISA plate reader (BioTek Instruments, Inc., Winooski, VT, USA) to determine protein expression levels.
4
Serum Cytokine Profiling
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Blood samples were transferred into micro-centrifuge tubes and allowed to clot at 4°C followed by centrifugation at 3000 × g for 5 min at 4°C. The supernatant pale yellow colored serum was pipette out carefully with the help of micropipettes into fresh micro centrifuge tubes, labeled and used for cytokine analysis. Serum from different groups were normalized to the protein content by Bradford method before the assay and levels of cytokines (IL-6, IL-10, IFN-γ and TNF-α) were determined by Sandwich ELISA according to the manufacturer’s instruction (Ray Biotech) in a Bio-Rad ELISA Reader.
5
Biomarker Monitoring After Surgery
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Blood samples were drawn from the central ear arteries, and sandwich ELISA (RayBiotech Life, Inc.) was used to detect C-reactive protein (CRP), interleukin-6 (IL-6) and FAP every two weeks after the operation according to the instructions.
6
Quantifying Metalloproteinase Levels via ELISA
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The expression of the MMP-2, MMP-7, MMP-9 metalloproteinase proteins and their TIMP-2 inhibitor were assessed using a sandwich ELISA (enzyme-linked immunosorbent assay) from RayBiotech. The ELISA test was performed three times for each slice. Laboratory procedures were carried out in accordance with the manufacturer's instructions. The first step was to perform the coat stage, which was carried out by adding solid phase to the wells (where there were specific antibodies to human proteins MMP-2, MMP-7, MMP-9 and TIMP-2), tested samples (tissue homogonates) and control samples. After the incubation process, the plate is washed. Secondary antibodies labeled with horseradish peroxidase conjugated streptavidin, were then added. The wells were rinsed again. In the next step, a substrate (tetramethylbenzidine, TMB) was added for the enzyme (horseradish peroxidase) bound to the antibody; as a result of the enzymatic reaction this turns into a coloured product. Using spectrophotometry, the colour intensity was determined after a specified duration of reaction (Thermo Labsystem Multiskan Ascent 354), thanks to which the measurement of the antigen concentration in the material used for the tests was obtained.
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