Thrombin-Antithrombin Assay Sample Clauses

Thrombin-Antithrombin Assay. The sample was centrifuged at 3040 g for double spin at seven minutes each spin, at room temperature and frozen at -40˚C within one hour of venepuncture. The Thrombin-Antithrombin Complex (TAT) Human ELISA Kit (Abcam) was used and the materials were supplied by the manufacturer. The reagents and working standards were prepared according to the manufacturer’s instructions, with various dilutions of the TAT standard prepared immediately before use. The TAT plasma samples were thawed in a water bath at 37˚C. Each sample was assayed in duplicate and each 96 well plate strip had 36 individual samples. The plate strip was annotated and a diagrammatic representation was labelled. 50 µl of TAT standard or plasma sample was added to each well and the bubbles carefully removed. The timer was started following the addition of the last sample, sealing tape was placed on the top of the xxxxx and the plate was left to incubate at room temperature for 2 h. The xxxxx were washed six times using a plate washer (ELX500 BioTek) and the excess water decanted. 50 µl of 1X Biotinylated TAT Antibody was added to each well and incubated at room temperature for 1h. The xxxxx were washed and 50 µl of 1X SP conjugate was added to each well and incubated for 30 min. 50 µl of Chromogen substrate was added to each well and incubated for 20 minutes or until optimal blue density. 50 µl of Stop solution was added and the absorbance was read from a microplate reader at 450 nm immediately. The ASCENT software calculated a standard curve and intra- and inter-assay coefficients of variability for every assay.