Serum TARC was measured using a double antibody sandwich ELISA (R&D Systems) as described previously (13 (link)).
Double antibody sandwich elisa
Manufactured by R&D Systems
Sourced in United States
The Double-antibody sandwich ELISA is a laboratory testing method used to detect and quantify specific proteins or other analytes in a sample. It involves the use of two different antibodies that bind to the target analyte, creating a 'sandwich' structure. This technique allows for the sensitive and reliable measurement of the target substance in the sample.
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11 protocols using double antibody sandwich elisa
1
Serum TARC Measurement by ELISA
2
Quantification of Serum BNP Levels
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The levels of serum brain natriuretic peptide (BNP) were quantified by enzyme-linked immunosorbent assay (ELISA) kits and performed according to the manufacturer’s instructions (R&D systems, Minneapolis, MN, USA) in accordance with the Double-antibody Sandwich ELISA instructions. The BNP values were calculated by standard curve method.
3
Quantification of Serum CHI3L1 Levels
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Serum CHI3L1 levels were determined by a double-antibody sandwich ELISA according to the manufacturer’s instructions (R&D systems, United States). Briefly, 96-well microplates were coated with 100 μL/well of the capture antibody (rat anti-human CHI3L1, 2.0 μg/mL) overnight at room temperature. After blocking with 3% BSA for 1 h, 100 μL of the patients’ serum (1:100 diluted in blocking buffer) was added and incubated for 2 h at room temperature. Subsequently, 100 μL/well of the detection antibody (biotinylated goat anti-human CHI3L1, 200 ng/mL) was added and incubated for 2 h at room temperature. Next, 100 μL/well of Streptavidin-HRP (1:200) was added and incubated for 20 min at room temperature. Finally, the substrate (tetramethylbenzidine) solution was added, and the reaction was stopped with 2 mol/L H2SO4 and read at an OD of 450 nm. Each test included a standard control (CV < 12%).
4
MIC-1 Quantification in Human Serum
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Plasma MIC-1 concentrations were measured using a double-antibody sandwich ELISA at room temperature (RT) as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Briefly, 96-well microplates (Costar, USA) were coated with mouse anti-human MIC-1 antibody (100 μL/well, 2.0 μg/mL) overnight. After blocking, 100 µL of the standard or serum (5-fold dilution in 3% BSA) were added and incubated for longer than 1 h. Hereafter, 100 µL of the biotinylated goat anti-human MIC-1 antibody (12.5 ng/mL) was added into each well and incubated for 2 h. Then, Streptavidin-HRP (100 μL/well) diluted to 200 times was incubated for 20 min. Finally, the substrate solution (tetramethylbenzidine) was added and the reaction was stopped using 2 N H2SO4. Absorbance was recorded at a dual-wavelength of 450/630 nm. Each plate also contained a standard control (coefficient of variation < 12%).
5
Quantification of Plasma CCL27 by ELISA
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Plasma CCL27 concentrations were measured using a double-antibody sandwich ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). In brief, 100 μl/well of the capture antibody (mouse anti-human CCL27, 4.0 μg/ml) was added to 96-well microplates overnight at room temperature. Test samples or CCL27 standard (100 μl/well) were added to the wells and the plates were incubated for 2 h. Detection antibody (biotinylated goat anti-human CCL27, 75 ng/ml) was added at 100 μl/well and the plate was incubated for 2 h. Finally, 100 μl/well of horseradish peroxidase-conjugated streptavidin was added to each well. After addition of a colorimetric reagent for 0.5 h, the reaction was stopped by the addition of 2 N sulfuric acid and the absorbance was measured at 450 nm. Each test included a standard control (coefficient of variation = 12%).
6
Serum Estrogen Measurement by ELISA
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Blood samples were collected and then serum was removed after centrifugation for 15 minutes at 1000 ×g. Plasma estrogen (E2) concentrations were measured using a double-antibody sandwich ELISA at room temperature as per the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was recorded at a dual-wavelength of 450/630 nm. Each plate also contained a standard control (coefficient of variation < 12%).
7
Serum Cytokine Quantification in Ischemic Rats
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At the end of the ischemia, blood was taken from rats in each group via the right carotid artery, and serum was separated through centrifugation at 1,006 × g for 15 min at 4°C. Double-antibody sandwich ELISA was employed for the detection of TNF-α, IL-6 and IL-1β levels in the serum, in accordance with the manufacturer’s instructions (R&D Systems, Inc.). Optical density values were determined by ELISA and the standard curve was drawn to calculate TNF-α, IL-6 and IL-1β concentrations in the sample.
8
Serum C3a and C5a Levels in COVID-19
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Sera were collected from mild or critical COVID-19 patients under the approval of the hospital ethics committee. Critical patients who were in the ICU were defined as having fever or suspected respiratory infection plus one respiratory rate >30 breaths/min, severe respiratory distress, or SpO2 < 90% in room air. Patients with pneumonia and no signs of severe pneumonia were defined as mild cases. Sera collected from 10 mild patients, and 18 critical patients were assayed. Sera from 10 healthy people were collected from the clinical laboratory. The serum C3a and C5a levels were detected by double antibody sandwich ELISA (R&D Systems). The serum C3a in some samples was not tested because of insufficient sample volume.
9
Monocyte Inflammatory Response Profiling
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Primary human monocytes were used to test the inflammatory response. The CD14 + human monocytes were isolated from buffy coats (National Blood Service, Cambridge, UK) as reported previously [37] (link) and seeded at a concentration of 1 × 10 5 cells per disc. Cytokine production was measured in the supernatant harvested at predetermined time points (6, (link)12, (link)24 and 48 hours). The levels of the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 were determined using a double antibody sandwich ELISA according to the manufacturer's protocol (R&D Systems). As a positive control, CD14 + human monocytes were treated with lipopolysaccharide (LPS) 10 µg mL -1 to evaluate the effect of a pro-inflammatory stimulus. Sample values were determined by comparison to a standard curve and all samples were assayed in triplicate.
10
Measuring Serum Inflammatory Cytokines
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Serum pro-inflammatory cytokines and mediators, including tumor necrosis factor-alpha (TNF-α), Interleukin-1-beta (IL-1β), and Interleukin-6 (IL-6) were determined using double-antibody sandwich ELISAs (R&D Systems, USA).
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