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INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, FEBRUARY 2009128
correlation (ICC) between the two methods was 0.82,again suggesting a good correlation. Individual valuesfor 30 samples analyzed by the two methods are givenin Table 1. The intra and inter assay coefficient ofvariation 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, ofwhich 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 ≥ 40yrs. The mean total antioxidant status in age category< 40 yrs was 0.987 ± 0.175 and 0.855 ± 0.187 mmol/lfor males and females, respectively. The observeddifference was statistically significant (p<0.001). In
the age group ≥ 40 yrs, the mean antioxidant was0.895 ± 0.25 and 0.996 ± 0.227 mmol/l for males andfemales, respectively. However, the difference wasnot statistically significant (p = 0.137) (Fig. 2).
Microplate assays are commonly used in themicrobiological assays and in food, drug and pharmaceutical industries, because of low sampleavailability. The assay provides the advantages of
reducing labor time, material cost and sample volume.Some of the assays have already been adapted formore convenient mass screening8,9, quantitativespectrophotometer assays10 as well as in agricultureand food industry11. A number of assays have beendescribed for estimation of total antioxidant status inserum, which are based on either of the techniques —spectrophotometric, fluorimetric or chemilumine-scence. Although adaptation of antioxidant assay onmicroplate has been reported and commercialized byCAYMAN, the method does not incorporate blankingof individual samples, which could result in
imprecision. Our method has a provision of blankingfor each and every set of samples analyzed.
The incubation time is very critical for theantioxidant assay and our microplate assay does notdeviate from the original protocol in terms ofincubation time. The microplate adaptation has also been reported by Wong et al12. The authors reported ahigh intra (4.3%) and inter (14.1%) assay. Theanalysis has been carried out with 2.5 µl serum bythese authors which could have resulted in the highvariation. The intra and inter assay coefficient of
Table 1—Total antioxidant status of samples (30) analyzed byRANDOX and microplate method
Serial no RANDOX method(mmol/l)
Microplate method(mmol/l)
1 0.676 0.772
2 0.734 0.743
3 0.792 0.861
4 0.871 0.842
5 0.800 0.711
6 0.705 0.801
7 0.672 0.651
8 0.600 0.563
9 0.631 0.744
10 0.722 0.715
11 0.681 0.782
12 0.753 0.843
13 0.782 0.851
14 0.813 0.785
15 0.870 0.860
16 0.700 0.730
17 0.672 0.653
18 0.740 0.730
19 0.682 0.662
20 0.733 0.741
21 0.874 0.841
22 0.601 0.562
23 0.722 0.713
24 0.630 0.744
25 0.675 0.77126 0.840 0.830
27 0.633 0.742
28 0.671 0.652
29 0.722 0.714
30 0.801 0.712
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
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SHORT COMMUNICATION 129
variation reported in our method compares well withthe original method.
The microplate method offers advantage over the
original protocol method. It reduces the assay timesignificantly. Sixty samples can be analyzed by thismethod in an hour as against only 18 samples by theoriginal method. Reduction of cost to one-fourthmaking it more amenable for research, is anotheradvantage that the plate method offers. The high cost per sample of the original RANDOX method makes itless amenable to widespread use.
Very little information on total antioxidant status isavailable from Indian population. Total antioxidantstatus in an industrial population of Baroda wasreported by Desai et al13 using ABTS method. Thereported mean total antioxidants were 1.8 ± 0.2mmol/l in healthy individuals. The mean valuesreported by our method are much lower, but since wehave not evaluated the micronutrient intake in oursubjects, we cannot comment on the values.
In conclusion, microplate adaptation of the ABTSmethod described here is cost-effective and would beuseful in epidemiological studies, where largenumbers of samples are to be analyzed.
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