Indian Journal of Biochemistry & Biophysics

Vol. 46, February 2009, pp. 126-129

Improved method of total antioxidant assay

Ruby Guptaa, Mukta Sharmaa, Ramakrishnan Lakshmya*,
Dorairaj Prabhakaranb and K Srinath Reddyc
a

Department of Cardiac Biochemistry,

c
Department of Cardiology,
All India Institute of Medical Sciences, Ansari Nagar,
New Delhi 110029
b
Center for Chronic Disease Control, Safdarjung
Development Area, New Delhi 110016
Received 06 October 2008; revised 20 January 2009
Commercially available analytical kits for the estimation of
total antioxidant status are expensive and time-consuming. Most
of the commercially available kits for total antioxidants estimation
are based on the principle of suppression of ABTS radical cation
formation by antioxidant in the serum sample. The method
requires stringent assay conditions, like exact incubation time and
the temperature (37C) of the reaction and on an average not more
than 40 samples can be analyzed on a day. We have adapted the
assay to a microplate, thereby allowing more number of samples
to be analyzed per day. Further, the reagent volume required is
one fourth than that for the original procedure thereby cutting
cost. Thirty samples were analyzed by original method on
spectrophotometer and our adapted microplate assay. The values
of total antioxidant obtained by the two methods correlated well.
Thus, total antioxidant can be estimated reliably using the
microplate method.
Keywords: Total antioxidant, ABTS, Microplate, Trolox,
Coefficient of variation.

Measurement of total antioxidant activity in body

fluids in patients with atherosclerosis, reperfusion
injury, septic shock and diabetes is of important
prognostic and diagnostic value. The multiple defense
system present in our body against damaging free
radicals is collectively called antioxidants. These
antioxidants are present in the serum and are
measurable. As measurement of individual
component of the antioxidants is difficult and timeconsuming, it is easier to measure total antioxidants
present in circulation. Various methods have been
reported for measuring total antioxidants in biological
fluids, such as the total peroxy radical trapping
______________
*
Corresponding author
Tel: 011-26594426
Fax: 011-26588663, 26588641
E-mail: lakshmy_ram@yahoo.com; lakshmyram@gmail.com

parameter assay1, the ferric reducing ability (FRAP)

method2, the phycoerythrin fluorescence-based assay3,
the enhanced chemiluminescence assay4, oxygen
radical absorbance capacity (ORAC)5 assay and the
trolox-equivalent antioxidant capacity (TEAC) assay6.
A high degree of imprecision poses a problem with
the oxygen radical absorbance assay. In phycoerythrin
fluorescence based assay, the decrease of fluorescence
is often not linear with time. The ORAC, TEAC and
FRAP are commonly used method for assessing total
antioxidant status. In the ORAC method, the oxidative
degradation of the florescent molecule phycoerythrin
is measured. The ORAC assay requires fluorescence
detector and takes a longer time to complete as
compared to the TEAC and FRAP assay. The FRAP
assay measures the ferric to ferrous reduction in
presence of antioxidants and is simple and relatively
inexpensive. The FRAP assay does not, however,
measure the thiol group containing antioxidants. The
TEAC assay is based on the principle of inhibition of
radical cation production by antioxidants in the
sample. The concentration of antioxidant in the
sample is inversely proportional to the absorbance of
the radical cation produced by 2,2-azino-bis-(3-ethyl
benzothiozoline-6-sulfonate) (ABTS) and ferrylmyoglobin. The TEAC assay has been commercialized by
RANDOX Laboratories. The method is expensive and
is limited by the fact that only four samples can be
analyzed at a time, as the incubation time of 3 min at
37C is critical for the assay.
A microplate adaptation of ABTS method has been
described previously by Wong et al12. The authors
reported a high intra and inter assay coefficient of
variation in the analysis and did not compare the
analytical performance with the original method.
Antioxidant assay on microplate reported and
commercialized by CAYMAN compromises on the
blanking,
thereby
affecting
the
analytical
performance. In this study, we have tried to adapt the
ABTS assay on a microplate which would help
cutting the cost to 25%, increase the number of
samples that could be analyzed per day and improves
on analytical performance, as compared to the
previously reported methods. The microplate method
described here has been compared with the method
reported by RANDOX in 30 samples. In addition, 161

SHORT COMMUNICATION

127

samples from an industrial population have been

analyzed for total antioxidant status using the
microplate assay method.

in absorbance between blank and sample (A blank A sample). Using this protocol, 60 samples could be
analyzed in 1 h.

Material and Methods

Comparison of the microplate method with the original

method

Total antioxidants assay using ABTS method

Blood was collected from 30 normal individuals.

Serum was separated and stored at -70C till the time
of estimation. Samples were analyzed for total
antioxidant status by both the methods. Intra and inter
assay CV was established for both methods by
running sample in triplicates in the same run and in
three different runs, respectively.

Total antioxidants were measured by the ABTS

method using kits from RANDOX as per
manufacturers instructions for comparison with the
microplate method. As per the method, metmyoglobin
(peroxidase) present in the chromogen provided in the
kit reacts with H2O2 to form ferrylmyoglobin, a free
radical species. The chromogen also contains ABTS
(2, 2-amino-di-[3-ethylbenzthiazoline sulphonate])
which reacts with ferrylmyoglobin to produce a
radical cation which has blue-green color and can be
measured at 600 nm. Antioxidants present in the
added serum cause suppression of this color
production proportional to their concentration.
Calibration of the assay was done using 6-hydroxy-2,
5, 8-tetra-methylchroman-2-carboxylic acid (trolox).
The results were expressed in mmol/l of trolox
equivalent. 20 l sample and 1 ml of chromogen were
required for the assay.
Modified assay on microplate

The additional requirement for the microplate

method was a multi-channel dispenser, a thermo
block to maintain the temperature at 37C and a plate
reader. The sample volume in the plate assay was
reduced to 5 l. Since the incubation time was critical
in the assay the reaction was carried out in one
column at a time. Blank, standard and control were
incorporated in each column. Since the sample
volume was only 5 l, the chromogen volume was
also proportionately reduced to 250 l. Plate was
incubated at 37C for 5 min on the thermo block and
absorbance read at 600 nm (initial absorbance -A1).
50 l of substrate kept at 37C was dispensed using
multi-channel dispenser, the stop watch was
immediately started and the plate was read again
exactly after 3 min (final absorbance -A2). The
calculation was done as per manufacturers
instructions. Difference in absorbance (A) was
calculated by subtracting A2 from A1 for blank,
standard and samples. Factor was calculated by
dividing the concentration of the standard by
difference in absorbance between blank and standard
(A blank A standard). Total antioxidant status
was calculated by multiplying the factor by difference

Analysis using microplate method

Blood samples were collected from 161 industrial

workers, who were recruited for a larger sentinel
surveillance study7. Total antioxidant status was
determined using the microplate method.
Statistical analysis

Correlation between the two methods (Pearson

correlation) and intra class correlation was computed
using SPSS. Paired t test was applied to observe the
difference between the two methods. Students t test
was applied to see difference between total
antioxidant levels in males and females industrial
workers.
Results and Discussion
The mean antioxidant status of the 30 samples
analyzed by the kit method and micro plate method
was 0.72 0.079 mmol/l and 0.74 0.080 mmol/l,
respectively. The total antioxidant status of the
samples estimated by the two methods correlated well
(correlation coefficient 0.70) (Fig. 1). The values
obtained by the two methods were not significantly
different by pairedt test (p = 0.129). Intra class

Fig.1 Scatter plot of serum antioxidant levels determined by

original RANDOX method and the microplate method

INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, FEBRUARY 2009

128

Table 1Total antioxidant status of samples (30) analyzed by

RANDOX and microplate method
Serial no
RANDOX method
Microplate method
(mmol/l)
(mmol/l)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

0.676
0.734
0.792
0.871
0.800
0.705
0.672
0.600
0.631
0.722
0.681
0.753
0.782
0.813
0.870
0.700
0.672
0.740
0.682
0.733
0.874
0.601
0.722
0.630
0.675
0.840
0.633
0.671
0.722
0.801

0.772
0.743
0.861
0.842
0.711
0.801
0.651
0.563
0.744
0.715
0.782
0.843
0.851
0.785
0.860
0.730
0.653
0.730
0.662
0.741
0.841
0.562
0.713
0.744
0.771
0.830
0.742
0.652
0.714
0.712

correlation (ICC) between the two methods was 0.82,

again suggesting a good correlation. Individual values
for 30 samples analyzed by the two methods are given
in Table 1. The intra and inter assay coefficient of
variation was 2.5% and 3.95% by the original kit
method, whereas it was 2.98% and 4.19%
respectively by microplate method.
For the study on total antioxidant levels in
industrial workers, 161 subjects were recruited, of
which 84 were males and 77 females. Among them,
44 males and 37 females were of < 40 yrs of age,
whereas 40 males and 40 females were of age 40
yrs. The mean total antioxidant status in age category
< 40 yrs was 0.987 0.175 and 0.855 0.187 mmol/l
for males and females, respectively. The observed
difference was statistically significant (p<0.001). In

Fig. 2 Distribution of total antioxidant levels in <40 years

(n = 44 males, 37 females) and 40 years (n = 40 males,
40 females) age group

the age group 40 yrs, the mean antioxidant was

0.895 0.25 and 0.996 0.227 mmol/l for males and
females, respectively. However, the difference was
not statistically significant (p = 0.137) (Fig. 2).
Microplate assays are commonly used in the
microbiological assays and in food, drug and
pharmaceutical industries, because of low sample
availability. The assay provides the advantages of
reducing labor time, material cost and sample volume.
Some of the assays have already been adapted for
more convenient mass screening8,9, quantitative
spectrophotometer assays10 as well as in agriculture
and food industry11. A number of assays have been
described for estimation of total antioxidant status in
serum, which are based on either of the techniques
spectrophotometric, fluorimetric or chemiluminescence. Although adaptation of antioxidant assay on
microplate has been reported and commercialized by
CAYMAN, the method does not incorporate blanking
of individual samples, which could result in
imprecision. Our method has a provision of blanking
for each and every set of samples analyzed.
The incubation time is very critical for the
antioxidant assay and our microplate assay does not
deviate from the original protocol in terms of
incubation time. The microplate adaptation has also
been reported by Wong et al12. The authors reported a
high intra (4.3%) and inter (14.1%) assay. The
analysis has been carried out with 2.5 l serum by
these authors which could have resulted in the high
variation. The intra and inter assay coefficient of

SHORT COMMUNICATION

variation reported in our method compares well with

the original method.
The microplate method offers advantage over the
original protocol method. It reduces the assay time
significantly. Sixty samples can be analyzed by this
method in an hour as against only 18 samples by the
original method. Reduction of cost to one-fourth
making it more amenable for research, is another
advantage that the plate method offers. The high cost
per sample of the original RANDOX method makes it
less amenable to widespread use.
Very little information on total antioxidant status is
available from Indian population. Total antioxidant
status in an industrial population of Baroda was
reported by Desai et al13 using ABTS method. The
reported mean total antioxidants were 1.8 0.2
mmol/l in healthy individuals. The mean values
reported by our method are much lower, but since we
have not evaluated the micronutrient intake in our
subjects, we cannot comment on the values.
In conclusion, microplate adaptation of the ABTS
method described here is cost-effective and would be
useful in epidemiological studies, where large
numbers of samples are to be analyzed.

129

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