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Reagents preparation: 1. For ABTS Reagent A dilute it with EtOH (for phenolic compounds and extracts) or PBS (for plasma) to an absorbance of 0.70 (±0.02) at 734 nm and 30 °C. Approximately a relation radical ABTS / EtOH 1 to 40. This solution is called ABTS Reagent A Solution. Keep it at -20 °C. 2. For standard solution preparation, add exactly 1 mL of deionized water to the ABTS standard vial. After that make the transfer of the 1 mL to a vial with higher capacity and fill up to 10 mL final volume. Once dissolved, keep it at -20 °C. Sample preparation: Dilute your sample in EtOH (for phenolic compounds and food extracts) or PBS (for plasma) such that, after introduction of 5 μL of each aliquot into 200 μL of ABTS Reagent A Solution, they produce between 5 % -35 % inhibition of the blank absorbance (ABTS•+ alone).
Standard Preparation Antioxidant activity is expressed as CEAC (Vitamin C equivalents antioxidant capacity). Prepare calibration curves in 1 mL tubes as shown in Table 1 (see kit Booklet). Keep it at -20 °C. Performing the assay:1.Add 5 μL of the sample or standard in each well. 2.Add 200 μL of ABTS Reagent A Solution previously diluted (see reagents preparation) in each well. 3.Mix the mixture at 30 °C for 4 minutes under continuous stirring. 4.Read the absorbance at 734 nm at 30 °C.
1.Calculate the absorbance at 734 nm as percentage of the absorbance of the uninhibited radical cation solution (Blank) according to the equation: Inhibition of A734 (%) = (1-Af/A0) x 100 Where A0 is the absorbance of uninhibited radical cation and Af is the absorbance measured 4 min after the addition of antioxidant samples. 2. Plot the inhibition of A734 of standards as function of their final concentrations (Table 1 in kit booklet). See Figure 2 (see images) for a typical standard curve. 3.Calculate the CEAC value of the samples using the equation obtained from the linear regression of the standard curve substituted of A734 values for each sample. CEAC (μM) = (sample inhibition A734- intercept)/slope