Peripheral blood samples were drawn a total of eight times: at baseline right before the exercise test, immediately after test termination, and once every hour until 6 h after the exercise test had terminated. Serum was extracted and stored at −80°C until analysis was performed. YKL-40 is stable in blood stored at −80°C 10 (link). Serum concentrations of YKL-40 were measured in November 2013. Samples were determined in duplicates by a commercial enzyme-linked immunosorbent assay (Quidel, Santa Clara, California, USA). The detection limit was 20 μg/l. The intra-assay coefficients of variation were 5% (at 40 μg/l), 4% (at 104 μg/l), and 4% (at 155 μg/l). The interassay variation coefficient of variation was less than 6%.
Enzyme linked immunosorbent assay
Manufactured by Quidel
Sourced in United States
Enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used to detect and measure specific proteins or other molecules in a sample. It utilizes antibodies and enzyme-mediated color changes to quantify the target analyte. ELISA is a widely used analytical tool in various fields, including clinical diagnostics, research, and quality control.
Automatically generated - may contain errors
Lab products found in correlation
Human complement factor b, by Complement Technology (2 mentions)
Human complement factor d, by Complement Technology (2 mentions)
Human complement c3 substrate, by Complement Technology (2 mentions)
Cyfra 21 1, by DRG International (1 mentions)
Protein g plus protein a agarose suspension, by Merck Group (1 mentions)
8 protocols using enzyme linked immunosorbent assay
1
Measuring Serum YKL-40 Levels After Exercise
2
Plasma YKL-40 Quantification Protocol
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Plasma samples were collected and handled according to standard operating procedures. YKL-40 concentrations in EDTA plasma were determined in duplicate, in samples stored at minus 80°C, by a commercial enzyme-linked immunosorbent assay (Quidel, Santa Clara, CA, USA). The detection limit was 15 µg/L. The intra-assay coefficient of variation (CV) was <5% and the inter-assay CV was <6%. All samples from each patient were analyzed on the same plate to reduce inter-assay CV.
3
Measuring Serum YKL-40 Levels
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Serum YKL-40 concentrations were determined by a commercial enzyme-linked immunosorbent assay (Quidel, Santa Clara, CA, USA). The intra-assay and interassay variations were 3.6% and 5.3%, respectively. The sensitivity of the assay was 20 ng/mL. The samples were measured in duplicate.
4
Inhibition of CVF-Bb Complement Activation
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Example 14
CVF-Bb complex is prepared from purified cobra venom factor (1 μM); human Complement factor B and human Complement factor D are available from a commercial source (Complement Technology, Tyler, Tex.). CVF-Bb complex at 3 nM concentration is incubated with test compound at various concentrations for 10 minutes at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human Complement C3 substrate (Complement Technology, Tyler, Tex.) is added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction is stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, is quantified by means of an enzyme-linked-immunosorbent assay (Quidel, San Diego, Calif.) and/or denaturing gel electrophoresis (SDS-PAGE). IC50 values are calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
5
Quantification of Pulmonary Biomarkers
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C4d-containing fragments were measured by an enzyme-linked immunosorbent assay (Quidel). This assay recognizes all C4d-containing fragments of activated C4 (C4b, iC4b and/or C4d), which together are referred to in this paper as C4d. Quantitative enzyme-linked immunosorbent assays were also used for the determinations of C4 (Assaypro), C5a (R&D), and CYFRA 21-1 (DRG International). BAL samples were diluted 1:10, 1:1000, 1:100, and 1:25 for the analysis of C4d, C4, C5a, and CYFRA 21-1, respectively. Sputum specimens were diluted 1:4 for C4d quantitation. Total protein was measured using the BCA protein assay (Pierce). All biomarker measurements were performed retrospectively by laboratory personnel not aware of the diagnosis. All procedures were carried out in a single laboratory following the manufacturers’ instructions.
6
Cobra Venom Factor Complement Assay
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Example 14
CVF-Bb complex is prepared from purified cobra venom factor (1 μM); human Complement factor B and human Complement factor D are available from a commercial source (Complement Technology, Tyler, Tex.). CVF-Bb complex at 3 nM concentration is incubated with test compound at various concentrations for 10 minutes at room temperature in PBS pH 7.4 containing 10 mM MgCI2 and 0.05% (w/v) CHAPS. Human Complement C3 substrate (Complement Technology, Tyler, Tex.) is added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction is stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, is quantified by means of an enzyme-linked-immunosorbent assay (Quidel, San Diego, Calif.) and/or denaturing gel electrophoresis (SDS-PAGE). IC50 values are calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
7
C5b-9 Depletion from Zymosan-Activated Serum
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C5b-9 was depleted in ZAS by immunoprecipitation as described by Ishikawa et al.38 (link). An anti-human C5b-9 monoclonal antibody (Quidel, San Diego, CA, USA) was added to freshly prepared ZAS at a molar ratio of 1:1 and incubated first at 22 °C (2 h), then at 4 °C (24 h) while the samples were continuously rotated. Protein G Plus/Protein A Agarose Suspension (Millipore, Billerica, MA, USA) was added to the mixture and incubated for 3 h at 22 °C with constant rotation of the samples. Substrate-bound immune complexes were removed from the serum by centrifugation (3,000 × g, 10 min) and filtration (0.8 μm). Sham samples of serum were prepared by identical methods except that the antibody was not added. SC5b-9 was quantified by enzyme-linked immunosorbent assay (Quidel) in the treated sera.
8
Serum YKL-40 Levels and Body Mass Index
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Venous blood samples were obtained during the morning (7-8 am) after an overnight fasting. These samples were separated by centrifugation and stored at -80°C. Serum YKL-40 levels were tested using an enzyme-linked immunosorbent assay (Quidel, San Diego, CA, USA). The body mass index (BMI) was calculated as weight in kilograms divided by height squared in meters (kg/m 2 ).
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