The objective of our study was to develop 96 well-microtiter plate 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) based decolorization assay method for analysis of large number of samples in a single run for the screening of antioxidant activity of various extracts such as food extracts, plasma antioxidants and synthetic antioxidants and optimization was carried out for parameters like concentration, incubation time, volume of the reagent and sample, reagent preparation, pH. 96 well-microtiter plate-ABTS based method denotes very good intra and inter assay analysis. IC50 values of standards and methanolic extracts of selected green leafy vegetables showed %coefficient of variation (CV) 4.1, 5 % for inter and intra-day assay results respectively was a significant achievement for the sample volume maintained at 5µL. The inter-day and intra-day accuracy were proved by the low values relative standard deviations (RSD) did not exceed 4.5%, indicates high reproducibility and precision of the method. This 96 well-microtiter plate-ABTS based decolorization assay method has advantages over the techniques like ferryl myoglobin/ABTS assay, such as no involvement of an intermediary radical, suppression of rapidly reacting antioxidants, low volume of sample, reaction at desired pH, and analysis of huge number of sample in a single run.

Keywords: ABTS, antioxidant activity, leafy vegetable, microtiter plate.