ICRISAT Archival Report 2006 - The seedlings of success in the ...

laboratory analysis for quantitative estimation of mycotoxins. For this purpose, we plan to develop plate-based,
tube-based, or filter paper-based (lateral-flow) assays that are suitable for on-site testing. Diagnostic reagents (polyand
monoclonal antibodies, mycotoxin standards, and enzyme conjugates) already developed for the mycotoxin
detection at ICRISAT will be used for the development of these tests. To achieve this objective, a simple ELISA
method is being developed to distinguish the variable concentration of aflatoxins (0, 2, 5, 10, and 25 ng ml -1 ) in the
samples based on color intensity (chromogenic reaction). ELISA plates were coated with 75 ng ml -1 of AFB1-BSA
and the AFB1 standard was added at 2, 5, 10, and 25 ng ml -1 , and the anti-aflatoxin rabbit antisera was added at 1:
25 K and 1: 50 K, and the alkalinephosphatase enzyme-conjugated anti-rabbit antibody was used at 1:4 000 and the
p-nitrophenylphosphate substrate was used at 0.5 mg ml -1 . Based on the color intensity in the wells of ELISA plate,
samples with more than 5 ng ml -1 toxin can be distinguished in 30 min. This qualitative ELISA test will aid in rapid
screening of samples to identify lots with more than 5 ng ml -1 toxin. This procedure is being refined further to
develop tube-based and filter paper-based assays.
2.5
2
OD at 405 nm
1.5
1
0.5
0- 4 ng ml -1
>5ng ml -1
0
0 ng 2 ng 5 ng 10 ng 25 ng 0 ng 2 ng 5 ng 10 ng 25 ng
10 min 20 min
concentration of aflatoxin B1 (ng/m l)
Fig. 1. Differentiation of aflatoxin contaminated lots based on color intensity in ELISA plate
Progress reported for 2007 for this activity will contribute to the milestones listed below.
Milestones: Tube/filter paper based semi-quantitative immuno assay developed for the on-site detection of aflatoxins
(FW/PLK) 2008
Milestones: Multiplex filter paper immuno assay developed for the rapid estimation of aflatoxins and fumonisins
(FW/PLK) 2009
Output target 6C 4: Aflatoxin resistant/tolerant groundnut genotypes identified
Activity 6C.4.1: Evaluate groundnut varieties for resistance to Aspergillus flavus and aflatoxin production by
in vitro inoculation studies and on-station testing in sick fields
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