TITLE: ABTS decolorization assay – in vitro antioxidant capacity
AUTHORS: Daniel C. Moreira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">This protocol describes how to perform the ABTS decolorization assay to assess potential in vitro antioxidant capacity of molecules and extracts using microtiter plates. Procedures are based on the method described in R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med. 26 (1999) 1231–1237.</div><style>
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[STEPS]
?. [ABTS radical preparation]
Prepare a 7 mM ABTS (e.g., A1888, Sigma-Aldrich) stock solution in ultrapure water.Example: Dilute 3.8408 mg in a final volume of 1,000 µL1.1Note that you will need 570 µL (190 µL for each replicate) of the final ABTS• stock solution per sample; 400 µL of the ABTS solution produces approximatelly 10,000 µL of ABTS• stock solution (see step 6).
?. Prepare a 245 mM APS (e.g., A3678, Sigma-Aldrich) solution in ultrapure water.Example: Dilute 5.5909 mg in a final volume of 100 µL.
?. [ABTS decolorization assay]
Pipet 10 µL of PBS (or other solvent used) plus 190 µL of ultrapure water into separate microtiter plate wells in triplicate. These will be used as reference/zero for the readings.
?. [Trolox standard solutions preparation]
Prepare a 2 mM Trolox (e.g., 238813, Sigma-Aldrich) stock solution in PBS3.Example: Dilute 1.0012 mg in a final volume of 2,000 µL.3If compatible, use the same solvent/buffer in which the sample to be analyzed is prepared to make the Trolox standard solutions.
?. [Calculation]
Using the average values of the triplicates for each standard concentration, calculate the decolorization effect caused by each samples/standard relative to the absorbance of the control (ABTS• + solvent) at 734 nm.Example:
?. Add APS to the ABTS solution so that the final APS concentration is 2.45 mM. This step is necessary to generate the ABTS radical (ABTS•).Example: Add 5.05 µL of APS to 500 µL of ABTS; final volume 505.05 µL.
?. Incubate it overnight (12–16 h) at room temperature in the dark.
?. Check the concentration of the ABTS radical (ABTS•) stock solution at 734 nm.Example: Dilute 10 uL of ABTS• solution in a final volume of 1,000 µL using ultrapure water; read it at 734 nm.
?. Prepare an ABTS• solution that absorbs ~0.700 at 734 nm.Example: If the solution prepared in the previous step absorbed 0.689, dilute 100 µL of the ABTS• stock solution in a final volume of 9,842.9 µL2. 2Remember that you will need 190 µL per well in the assay; each sample/control/standard is analyzed in triplicate (3 wells).Example: If you are going to assess two samples at four concentrations each, build a standard curve with six concentrations, and read a blank reaction control (all in triplicate), you will need 8,550 µL of the ABTS• stock solution (absorbing ~0.700).
?. Check the absorbance at 734 nm, adjust if necessary, and store until use.Example:  If the solution prepared in the previous step absorbed 0.805, diluted it 1.15-fold (9,000 µL in  a final volume of 10,350 µL) to achieve an absorbance of ~0.700.
?. [Sample preparation]
Dilute5 the samples at the desired concentrations6.5If a solvent other than PBS is necessary to dilute the sample, prepare adequate solvent controls in the next step. Example: If you need 50% (v/v) DMSO to dilute your sample, also assess 10% DMSO (without sample) solution in the assay to check for any interference.6For peptides, we usually use 2.0, 1.0, 0.5, and 0.25 mg/mL concentrations. Note that for peptides with strong activity (i.e., 100% ABTS• scavenging), we further dilute them to 0.125–0.031 mg/mL.
?. [Trolox standard solutions preparation]
Prepare several Trolox solutions at concentrations from 12.5 to 400 µM4 using PBS in Eppendorf tubes.Example: First, dilute the 4 mM stock by 5-fold to 400 µM, and dilute it serially to 200, 100, 50, 25, and 12.5 µL (e.g., 100 µL of the previous concentration + 100 µL PBS). 4Under the conditions described in this protocol, the change in absorbance at 734 nm is linear within the 12.5–400 µM Trolox range (see Calculation section).
?. [ABTS decolorization assay]
Pipet 10 μL of each trolox standard solution (including the control7, solvent only) and each sample dilution into separate microtiter plate wells in triplicate.7These are the reference wells of maximum ABTS• concentration, in which no sample or standard were added.
?. [ABTS decolorization assay]
Add 190 µL of the ABTS• solution prepared in Step 7 into each well.
?. [ABTS decolorization assay]
Mix the wells content using the 'shake' function of the microtiter plate reader, incubate it for 5 minutes in the dark and read it at 734 nm.
?. [Calculation]
Build a standard curve: plot trolox concentrations in mg/mL on the X-axis and decolorization (%) on the Y-axis. Calculate a linear regression (Y = a × X + b; e.g., 'add a linear trendline' in Microsoft Excel).Example:
?. [Calculation]
Calculate sample's antioxidant capacity relative to that of trolox using the equation generated in the previous step.Example: