TAC, in terms of copper reducing equivalent (CRE) of the sample, was evaluated using OxiSelect™ TAC Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA; Catalog Number: STA- 360) [25 (link)]. The components of the kit were uric acid standard (Part No. 236001), reaction buffer, 100X (Part No. 236002), copper ion reagent 100X (Part No. 236003) and stop solution, 10X (Part No. 236004). The assay protocol was as per the manufacturer’s product manual. Briefly, 20 μL of the sample in various concentrations and 180 μL of 1X reaction buffer were transferred to each well in a 96 well plate and mixed thoroughly. An initial absorbance was taken at 490 nm. The reaction was started by adding 50 μL of 1X copper ion reagent into each well and incubated on an orbital shaker for 5 min. Then the reaction was stopped by adding 50 μL of 1X stop solution to each well and absorbance was measured again. The net absorbance was calculated by subtracting the initial reading from the final reading and the mM uric acid equivalent (UAE) was determined from the uric acid standard curve. Finally, the CRE was determined by multiplying UAE by 2189.
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