New diagnostic tools

Quantitative detection of nucleopolyhedroviruses of pod borers

To compliment our ongoing NPV production activities, we produced polyclonal antibodies against polyhedra protein of Helicoverpa armigera nucleopolyhedrovirus ( Ha NPV), Spodoptera litura nucleopolyhedrovirus
( Sli NPV), and Amsacta albistriga nucleopolyhedrovirus ( Ambl NPV), to develop an ELISA for diagnosis and quality control of the NPVs. Polyocclusion bodies (POBs) were purified from NPV infected insect cadavers and used for the purification of polyhedrin protein by alkali treatment, followed by differential centrifugation and isoelectric focusing. The purified polyhedrin protein was used as antigen to produce antibodies in rabbits. The specificity, sensitivity and titer of the antisera were assessed by direct-antigen coating ELISA and western immunoblotting. The polyclonal antibodies did not react with the insect protein extracts revealing high specificity of the antibodies, and the antibody titer in various bleeds ranged from 1:2,000 to 1:40,000 for detecting respective polyhedrin proteins. In addition, the antibodies of each virus cross-reacted with polyhedrins of other viruses in the study, indicating high conservation in polyhedrin protein among the three species of NPVs. The detection limits of antiserum in indirect ELISA was 20 nanograms(ng) of polyhedrin in case of Ha NPV and Sli NPV, whereas 8 ng in case of Ambl NPV. Further work is continuing towards quantification of polyhedrin for the early diagnosis of infection progress in the field, as well as quality control of NPV to accurately quantify the virus titer in the formulations.

ELISA for quantitative estimation of Abscisic acid – a tool for screening for drought resistance

Abscisic acid ( ABA ) concentration in plants can affect plant response to drought and has been suggested as a selection criterion to improve drought tolerance. Several studies attributed increase in ABA concentration to reduction in stomatal conductance and also increase in root and shoot biomass. In order to assist selection of genotypes for high ABA, it is necessary to have a tool that can provide an estimate of ABA levels in the test plants. This activity was undertaken with a purpose ofdeveloping an ELISA test for quantitative estimation of ABA in plants. ABA ( ± cis/trans)-BSA conjugate was synthesized in the laboratory and used as immunogen for producing polyclonal antibodies in a rabbit. Polyclonal antibodies were characterized for sensitivity and specificity, and they were used to develop an indirect competitive ELISA (cELISA) for measuring ABA. A simple, cost-effective and rapid procedure for extracting free ABA from leaf samples was standardized, and ELISA for measuring ABA was established. The method was validated by spiking known amounts of ABA. Detection limits of 5 picograms/ml ABA was determined with the antibodies diluted to 1:30,000. The recovery of ABA spiked in extracts was 94±6%. The method was applied to the measurement of ABA in groundnut samples using varieties TMV 2 (drought susceptible), TAG 24 (drought tolerant), ICGS 44, ICGS 76 and ICGV 86031, under variable water volume treatments. Samples from controls and water-stressed plants were harvested at weekly intervals for three weeks; free ABA was extracted and analyzed in ELISA. Rate of transpiration efficiency (TE) was also measured simultaneously. ELISA results revealed relatively high ABA (twice that of control) in water-stressed TAG 24 and ICGV 86031, compared to controls. In TMV 2, ICGS 44 and ICGS 76, ABA levels were marginally low in stressed plants compared to controls. The data correlated with TE and physical observations, indicating that high ABA concentration in TAG 24 and ICGV 86031 correlated with tolerance to water stress. This validation suggests that the simple ELISA test can be used to screen large numbers of samples in a relatively short time for ABA levels (40 samples can be processed per day; with US$2 per sample analysis). Current studies focus on validating this test by analyzing a large number of groundnut and pearl millet samples.

Diagnostic test for aflatoxin estimation in humans

A simple competitive ELISA (cELISA) was developed for the estimation of aflatoxin biomarkers in human serum. This test is based on the estimation of AfB 1 -lysine, a metabolite of AfB1, whose concentration in the blood albumin fraction has been shown to correlate with dietary aflatoxin intake over the previous 2 to 3 months and the level of DNA damage in the liver. Synthetic AfB 1 -lysine-BSA was produced in the laboratory and used as immunogen to produce the polyclonal antibodies in a rabbit at ICRISAT. These antibodies were characterized for specificity (check for any non-specific reactions), sensitivity (lowest limit of detection of target molecule) and titer (maximum usable dilution of antiserum), and were then used to develop a cELISA to measure AfB 1 -biomarker. A simple procedure was developed to isolate albumin fraction from blood samples and its hydrolysis with proteinase K. Hydrolyzed albumin was used in cELISA to assess the biomarker levels. The current test can detect levels of AfB 1 -lysine in blood as low as 5 picograms (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 140 blood samples from the Hyderabad region in India. The results revealed that almost 10-20% of the samples contained AfB 1 -lysine in the range of 5 - 30 pg mg -1 albumin, indicating that there is a substantial exposure to aflatoxins through diets. 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 latest test for monitoring ‘aflatoxin exposure in humans' compliments our low-cost diagnostic test developed earlier for the detection of ‘aflatoxins in agriculture commodities'. Both of these tests now allow conducting epidemiology studies to identify aflatoxin-exposed populations, determine the source of contaminated food and stimulate appropriate management approaches to limit dietary-aflatoxin exposure, thereby enhancing the food and human health safety, and reducing the risk of liver cancer.

For further details contact :Farid Waliyar