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

Milestone: Two best transgenic groundnut events with resistance to A. flavus used for introgression into locally
adapted groundnut genotypes and their evaluation (KKS/FW/PLK/SNN) 2011
Output target 6C.3: Simple and cost-effect test for the estimation of mycotoxins (Aflatoxins, Fumonisins, and
Ochratoxin-A) in crops and commodities, and aflatoxin-adducts in human serum developed and validated
Activity 6C.3.1: Develop a diagnostic test to determine the human exposure to aflatoxins
Milestone: Enzyme-linked immunosorbent assay (ELISA) assay developed for the estimation of aflatoxin adducts in
human serum (FW/PLK) 2007
Development of ELISA for determining human exposure to aflatoxins: Aflatoxins are naturally produced foodborne
metabolites of Aspergillus flavus and related fungi found in most of the food crops grown in tropics and subtropics.
Aflatoxins constitute a group of four compounds, aflatoxin B1 (AfB1), B2, G1, and G2. Severe intoxication
due to consumption of highly contaminated food results in acute liver damage and even death. Frequent exposure to
sub-lethal doses leads to several nutritional and immunological consequences and greatly increases the risk of liver
cancer. Moreover, research during the past two decades has established a synergistic interaction between Hepatitis B
virus infection and AfB1 in causing liver cancer. Various studies suggest that the chronic type of aflatoxin poisoning
is common in many parts of developing countries in Asia and Africa, and usually goes unnoticed. In order to assess
the risk of human exposure to aflatoxins, a simple competitive enzyme-linked immunosorbent assay (ELISA) was
developed. This test is based on the estimation of AfB 1 -lysine, a metabolite of AB1, whose concentration in the
blood albumin fraction has been shown to correlate with dietary aflatoxin intake over the previous 2 - 3 months and
the level of DNA damage in the liver. The test involves isolation of the albumin fraction from blood, followed by
digestion of albumin and estimation of AfB 1 -lysine content by ELISA using AfB1-lysine polyclonal antibodies
produced at ICRISAT. These polyclonal antibodies were raised against AFB1-lys-BSA adducts in a New Zealand
White rabbit. In the ELISA test, polyclonal antibodies are first bound to the AfB 1 -lysine present in the extracted
albumin. Then, the antibody- AfB 1 -lysine complex is detected using an alkaline phosphatase enzyme-labeled
reporter antibody. Finally, the enzyme-labeled reporter antibody is detected using a colorimetric reaction that
provides an estimate of the original AfB 1 -lysine concentration. The current test can detect levels of AfB 1 -lysine in
blood as low as 5picogram per milligram (pg mg -1 ) albumin. The test is simple to perform, inexpensive, and is
effective for routine monitoring of human as well as animal samples for aflatoxin exposure.
The assay was used in a survey of 80 HBV positive blood samples from the Hyderabad region in India. The results
revealed that almost 20% of the samples contained AfB 1 -lysine in the range of 5 - 30 picogram mg -1 albumin,
indicating that there is a high risk for liver cancer in Hepatitis B positive individuals. This simple test can be used to
screen large numbers of samples and provides scope for preventive interventions in individuals at high risk of liver
cancer. This test compliments commercial HBV testing, and the ELISA test we developed earlier for the detection of
aflatoxins in foodstuffs. Both tests allow field studies to identify aflatoxin-exposed populations, determine the
source of contaminated food, and initiate management approaches to limit dietary-aflatoxin exposure, thereby
enhancing the food and human health safety and reducing the risk of hepatocellular carcinoma.
Lava Kumar/Farid Waliyar/CN Reddy
Activity 6C.3.2: Develop simple and cost-effective assays for the detection of mycotoxins in crops and
commodities
Milestones: Simplified ELISA-based assay for the detection of mycotoxins developed (FW/PLK) 2007
Detection of food-borne mycotoxin contaminants is essential to ensure food safety. A simple and cost-effective
competitive ELISA technique has been developed at ICRISAT for the detection of aflatoxins (AfB1, AfB2, AfM1,
and total toxins), ochratoxin A ,and fumonisins; which are being widely used. However, these laboratory-based
techniques require skilled technicians, and samples need to be sent to the laboratory for testing purpose. However,
simple on-site mycotoxin detection methods would aid in testing large numbers of samples by non-experienced
technicians in the fields, and rapid identification of contaminated lots, which can be segregated and sent for
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