ELISA kits with colorimetric output were used to determine the plasma concentrations of PAI-1 (ab184863, Abcam), fibrinogen (ab241383, Abcam), plasminogen (ab108893, Abcam), tissue plasminogen activator (t-PA) (ab190812, Abcam), platelet factor 4 (PF4) (ab189573, Abcam), interleukin (IL) 1 receptor-like 1 (ST2) (ab254505, Abcam), von Willebrand factor (vWF) (ab223864, Abcam), and tryptase (EKU10581, Biomatik) according to the manufacturer’s instructions. The plates were read at 450 nm using an automated microplate reader (Biotek Epoch). For each subject, we report the mean value from at least two replicate determinations with a coefficient of variation (CV) lower than 20%. The mean intra-assay CV was lower than 10% for all biomarkers and the inter-assay CVs were 11% for PAI-1, 10% for fibrinogen, 7% for plasminogen, 8% for t-PA, 9% for PF4, 14% for ST2, 10% for vWF, and 12% for tryptase. The plasma concentrations of each biomarker were determined using a standard curve, constructed and fit using a 4P logistic regression equation, and the assay values were corrected by the appropriate dilution factor in each case.
Ab223864
Manufactured by Abcam
Ab223864 is a laboratory equipment product. It is a device used for the measurement and analysis of various biological samples.
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3 protocols using ab223864
1
Multiplex Biomarker Quantification in Plasma
2
Comprehensive VWF Evaluation Protocol
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We used multiple assays to comprehensively evaluate VWF, including: 1) VWF antigen (VWF:Ag) and VWF propeptide (VWF:pp) measured by enzyme-linked immunosorbent assay (Cat #: ab223864 [Abcam] and CS-MW1939 [Cell Sciences]); 2) thiols exposed on the surface of VWF multimers (Supplemental Methods ); 3) ristocetin-induced platelet agglutination (RIPA) (Helena Laboratories); 4) VWF binding to collagen (VWF:CB) (Supplemental Methods ); 5) platelet adhesion under flow conditions (Supplemental Methods ); 6) VWF multimers using nonreducing sodium dodecyl sulfate–agarose gel electrophoresis (Supplemental Methods );27 (link) 7) shear-induced platelet aggregation (SIPA) (Supplemental Methods ); 8) the exposure of the A1 domain on VWF using immunoprecipitation (Supplemental Methods ); 9) endothelial permeability (Supplemental Methods ); and 10) ADAMTS-13 antigen (ADAMTS-13:Ag) using enzyme-linked immunosorbent assay (Cat #: ab234559; Abcam) and cleaving an exogenous VWF peptide measured using a FRET (fluorescence resonance energy transfer) assay (Diapharma).
3
Endothelial Dysfunction Biomarker Assay
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