A sandwich ELISA was performed to determine the candidate protein levels of S100A9, MUC5AC, TGF-β1, and angiopoietin-2. Quantitation of these proteins in sera of CCA patients and non-CCA patients were compared with the normal ultrasonography group by using a Quantikine ELISA Kit (S100A9, CSB-E11834h, Cusabio, Houston, TX, USA; MUC5AC, CSB-E10109h, Cusabio, Houston, TX, USA; TGF-β1, DB100B, R&D systems, Minneapolis, MN, USA; angiopoietin-2, DANG20, R&D systems, Minneapolis, MN, USA). According to the manufacturer’s instructions, the plate that was coated with primary antibody specific to each protein, was added to assay diluent to each well. Standard, control, and diluted samples were added to each well in duplicate and they were incubated for 2–2.5 h at room temperature with gentle shaking. After washing, the biotinylated antibody specific for each candidate protein was added for 1–2 h’ incubation time. Subsequently, streptavidin-HRP solution was added to each well for 45–60 min at room temperature. The TMB substrate solution was added and was protected from light. The reaction was stopped with hydrochloric acid and the plates were read on an ELISA reader using Magellan at the optical density (OD) of 450 nm. The results were calculated by reference to the standard curve that related to the concentration in each protein.
Dang20
Manufactured by R&D Systems
Sourced in United States
DANG20 is a recombinant protein that functions as a cytokine. It is produced in a human cell expression system.
Automatically generated - may contain errors
Lab products found in correlation
11 protocols using dang20
1
Quantitative ELISA Analysis of Biomarkers
2
Quantification of Ang2 and IGF1 in Serum and Ascites
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Serum and ascites were collected from patient cohorts. The Ang2 and IGF1 levels were detected by ELISA using human Angiopoietin2 (DANG20, R&D Systems) or human IGF1 Quantikine ELISA kit (DG100, R&D Systems).
3
Cerebral Malaria Retinopathy and Biomarkers
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This study is nested in a published study whose objective was to understand the value of malarial retinopathy in cerebral malaria21 (link). Participants included children admitted to Kilifi County Hospital (formally known as Kilifi District Hospital) between July 2005 and December 2011 with a combination of malaria parasites and coma. This means all the children in this study had severe malaria. Retinopathy status was assessed at admission as described previously21 (link). In this study we included all those children who were positive for malaria and had a parasite sample frozen in TRIzol available for var expression analysis. Plasma PfHRP2 concentration was determined as described in Kariuki et al.21 (link). Angiopoietin-2 and soluble ICAM-1 (sICAM-1) plasma levels were determined using commercial ELISA kits DANG20 and DY720 respectively from R&D following manufacturer’s protocol.
4
ANGPT2 Protein Quantification in Cell Culture and Serum
5
Measurement of Antibody Levels to Infected Erythrocyte Surface
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Rosette frequency data was obtained as described previously [10 ]. IE surface antibodies data was obtained as described in [10 ]. Briefly we measured each participant’s IgG antibody levels (acquired as mean fluorescent intensity (MFI)) to the infected erythrocyte surface of eight ex vivo clinical isolates grown to the trophozoite stage using flow cytometry. The eight isolates were selected on the basis of their var expression (ranging from high to low cys2 expression). The median MFI value of this IE surface recognition by IgG against the eight isolates was calculated for each participant [10 ].
Ang-2 level in the acute plasma was determined by a commercial ELISA Kit from R & D (cat no; DANG20) using the manufacture’s protocol.
Ang-2 level in the acute plasma was determined by a commercial ELISA Kit from R & D (cat no; DANG20) using the manufacture’s protocol.
6
Quantifying Angiopoietin Levels in Plasma
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To detect the levels of angiopoietin-1 and angiopoietin-2 in plasma, enzyme-linked immunosorbent assay (ELISA) kits were used and carried out according to the manufacturer’s protocol (DANG10 and DANG20, R&D, MN, USA). Briefly, standards of angiopoietin-1 and angiopoietin-2 were prepared, and 1:5 diluted plasma samples were pipetted into wells with specific monoclonal antibody pre-coated microplate. After washing away unbound solution, enzyme-linked monoclonal antibodies were added to wells. Following a wash to remove any unbound antibody-enzyme solutions, a substrate solution was added to wells and the color developed in proportion to the control or sample concentrations. Finally, a tetramethylbenzidine solution was added to stop the reaction. We detected the intensity of the reaction color at 450 nm wavelength with a microplate reader (BioTek Instruments, VT, USA). All measurements were performed in duplicate.
7
Quantifying Human Angiopoietin-2 via ELISA
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Human Angiopoietin-2 Quantikine ELISA (DANG20; R&D Systems) was used according to the manufacturer’s instructions. Details can be found in Supplementary Material . The quality control measures of % recovery mean and % coefficient of variation were within acceptable ranges, and the interassay coefficient of variation was <4.0%.
8
Plasma Biomarkers in Clinical Study
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All routine lab tests were performed at the hospital’s clinical chemistry department. ELISA’s were used to measure the plasma protein concentrations for ANGPT2 (DANG20, R&D Systems, Minneapolis, MN, USA), ANGPT1 (DANG10, R&D Systems, Minneapolis, MN, USA), von Willebrand factor (ab108918, Abcam, Cambridge, UK), and ADAMTS13 (ab234559, Abcam, Cambridge, UK), according to the manufacturer’s instructions. Data for thrombin–antithrombin (TAT) complexes were available for some patients from a previous study [10 (link)].
9
Plasma Biomarker Measurement Protocol
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10
ELISA Quantification of Plasma Biomarkers
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Plasma sPLA-2 Type IIA, Ang1 and Ang2 was measured by ELISA kit (Cayman Chemical: #501380; R&D Systems: #DANG10, #DANG20, respectively), as per manufacturer instructions. Samples were run in duplicate with absorbance read at 450 nm in real time using a spectrophotometer. sPLA-2, Ang1 and Ang concentrations were generated from a seven-point four-parameter logistic standard curve.
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