For detection of FIX protein, the F9 monoclonal antibody (M01), clone 2C9 (Abnova) was employed in a dilution of 1:500. GAPDH protein expression was detected using GAPDH antibody (1:2000; Cell Signaling Technology). Detection was performed via licor Odyssey infrared imaging system. Recombinant FIX (BeneFix, Pfizer) was employed as positive control. Supernatant obtained from CHO cells was employed for ELISA-based quantification of FIX antigen (FIX:Ag) using ZYMUTEST Factor IX from HYPHEN BioMed according to the manufacturer’s protocol including a standard FIX calibrator concentration set to 100% which equals 100 U/dl. Thus, FIX:Ag levels are expressed as % FIX protein. FIX activity was determined using a one-stage coagulation assay. Samples were diluted 1:5 in imidazol buffer and mixed with an equal amount of FIX deficient plasma and Actin FS APTT reagent (all from Siemens Healthcare). After incubation at 37°C for 2 min, 0.025 M calcium-chloride was added, and the coagulation time recorded in an Amelung KC10 coagulometer. Calibration curves were generated with human standard plasma diluted in FIX deficient plasma.
Actin fs aptt reagent
Manufactured by Siemens
Sourced in Germany
The Actin FS aPTT reagent is a laboratory equipment product used for the determination of the activated partial thromboplastin time (aPTT) in human plasma. It is a reagent that initiates the intrinsic pathway of the coagulation cascade, allowing for the assessment of the overall function of the intrinsic and common coagulation factors.
Automatically generated - may contain errors
4 protocols using actin fs aptt reagent
1
Quantitative FIX Protein Analysis
2
Emicizumab Concentration Measurement Protocol
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The emicizumab concentration was measured with the mcOSA on a Sysmex CS2500, a coagulation analyzer (TOA Medical Electronics Co., Ltd., Hamburg, Germany) with Actin FS aPTT reagent (Siemens, Marburg, Germany). Standard dilutions for CS2500 were applied and were followed by an extra dilution 1:8 with Owren’s Veronal Buffer (calcium system buffer) to minimize FVIII interference, then FVIII‐deficient plasma, Actin FS, and CaCl2 were added (Siemens, Marburg, Germany). Emicizumab concentrations were deduced from an emicizumab calibration curve, based on the plasma calibrator (r2 Diagnostics, South Bend, IN, USA; catalog #152‐401‐RUO, 102 µg/mL, lot no. EC0140). The plasma controls (r2 Diagnostics; catalog no. 152‐401‐CE) of level 1 (26.6 µg/mL; lot no. E10310) and level 2 (73.4 µg/mL; lot no. E20410) were used as internal quality controls. The calibration curve was linear over a concentration range of 10 to 200 µg/mL with an R2 of 1.00. The within‐run and between‐run precision (relative standard deviation [RSD], %) of the control samples ranged between 3.5% and 5.7%. The RSDs of the two control samples were similar after four freeze‐and‐thaw cycles. The LLOQ was 2 µg/mL.
3
Factor VIII Activity Determination
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Factor VIII activity was determined by an aPTT based one-stage assay, using appropriate factor deficient plasma and Actin FS aPTT reagent (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany).
4
Coagulation and Inflammatory Markers in Sepsis
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Standard coagulation markers (aPTT, PT and fibrinogen) were determined using a Sysmex CA1500 automated analyser within 2 hours of collection. Platelet count was determined using a Sysmex XE 2100 automated haematology analyser within 2 hours of collection. Factor VIII activity was determined by an aPTT based one-stage assay using appropriate factor deficient plasma and Actin FS aPTT reagent (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). D-dimer concentration was determined using TriniLIA Auto-D-dimer® turbidimetric assay.
Inflammatory markers were determined to quantify the inflammatory response at each stage of the sepsis spectrum and were selected based on previous studies examining the inflammatory effects of sepsis. Concentration of each of the inflammatory markers was measured in platelet poor plasma using enzyme-linked immunosorbent assay (ELISA) kits for PCT, TNF-α, IL-6 and sE-Selectin. The standard methodology was followed as supplied by manufacturer.
Inflammatory markers were determined to quantify the inflammatory response at each stage of the sepsis spectrum and were selected based on previous studies examining the inflammatory effects of sepsis. Concentration of each of the inflammatory markers was measured in platelet poor plasma using enzyme-linked immunosorbent assay (ELISA) kits for PCT, TNF-α, IL-6 and sE-Selectin. The standard methodology was followed as supplied by manufacturer.
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