Aflatoxin contamination is widespread in staple crops like peanut, maize, sorghum, pearl millet, chillies, pistachio, cassava etc., and compromises the safety of food and feed supplies. It is important to be able to detect and quantify aflatoxins in commodities to protect human and animal health. Many different methods, including antibody-based ones, are available for quantitative estimation of aflatoxins. However, most of these methods such as HPLC, HPTLC, and TLC are expensive and/or difficult to use in developed countries. Using the state-of-the-art facilities at ICRISAT, we developed polyclonal and monoclonal antibodies for the detection of total aflatoxins, aflatoxin B1 and M1 (secreted in milk). These were used to develop a simple and inexpensive competitive enzyme-linked immunosorbent assays (cELISA) that has lower detection limits (1.0 µg/kg) and cost (about $1 per sample) less than other available methods. More than 100 samples can be analyzed in a day. These tests have provided a unique opportunity for ICRISAT and its partners to conduct field studies to select resistant genotypes, identify high risk populations and determine the dietary sources to stimulate appropriate interventions to enhance the food and human health safety, trade and thereby farmers' income.
Aflatoxins are the toxic secondary metabolites produced by
There are several chemical methods available for detection and estimation of aflatoxin, including high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and mini-column methods. However, most of these methods are expensive, laborious, time consuming, and require extensive sample cleanup (
Cross reaction and minimal inhibition observed with aflatoxin B1 (AFB1) monoclonal antibodies.
The distinguishing feature of the competitive assay format is that the combination of an unknown amount of analyte introduced from the sample and the reference analyte compete for binding to a limited number of antibody binding site. This assay can be performed with either the analyte or the antibody adsorbed to the solid phase. Two types of cELISAs have been developed for the analysis of aflatoxins, and both types are heterogeneous assays which produce the uniform results. Direct ELISA involves the use of an aflatoxin-enzyme conjugate, whereas indirect ELISA involves protein-aflatoxin conjugate and a second antibody to which the enzyme has been conjugated (
In the indirect competitive ELISA, commercially available AFB1-BSA conjugate was coated on to the wells of a microtiter plate. Later, aflatoxin standards, as well as samples with specific rabbit antibodies, were added to the plate before incubating it at 37 C for 1 hr. After washing the plate to determine the amount of antibody bound in the wells, goat anti-rabbit IgG labeled with alkaline phosphatase was added followed by addition of p-nitrophenyl phosphate substrate. Thus, toxin in the sample or standard and toxin bound on the well surface competes for the binding site on the specific antibody in the solution (
Recovery of aflatoxin B1 (AFB1) by indirect competitive ELISA from groundnut samples spiked with different concentrations of toxin.
Comparison of analysis of aflatoxin B1 (AFB1) concentration in naturally contaminated chilli samples by indirect competitive-ELISA and HPLC methods.
Recovery of aflatoxin M1 (AFM1) from artificially contaminated milk samples as determined by ELISA.
In the direct competitive assay, specific antibodies are first coated on to a high quality high binding microtiter plate. Then the sample solution or various concentrations of standard toxin are generally incubated simultaneously with the enzyme conjugate in the ELISA plate. After appropriate washings, the amount of enzyme bound to the plate is determined by incubation with suitable substrate (p-Nitrophenyl phosphate for the alkaline phosphatase enzyme). At ICRISAT, a different kind of conjugate was prepared using commercially available AFB1-BSA which was conjugated to enzyme alkaline phosphatase or penicillinase or horse radish peroxidase and used in the direct ELISA instead of AFB1-enzyme conjugate (
In both the cELISAs, after addition of the substrate, the resulting color development is then measured spectrophotometrically at 405 nm using an ELISA reader to record the optical density values for standards as well as unknown samples. To calculate the aflatoxin content in the unknown samples, a regression curve using AFB1 standard's optical density (OD) values was drawn, and based on the regression equation the AFB1 in the samples was determined (
Linear regression curve and equation for aflatoxin standard in cELISA.
The application of mycotoxin immunoassays is not limited to foods and feeds; it has been used as a sensitive approach for monitoring of mycotoxins in body fluids and tissues and organs of humans and animals that have been exposed to the mycotoxins. Currently we are developing immunoassays to detect the aflatoxin in human and animal blood. This will not only be helpful in detecting aflatoxin in the human blood but also for monitoring aflatoxicosis in human beings and livestock (
One of the common challenges of immunoassay for food analysis is matrix interference causing false positives. Because there is a high probability of the presence of structurally related compounds in the sample that may react with the antibody, the sample matrix should be tested before the assay. This occurs when (a) the enzyme activity is inhibited by the presence of interferences in the sample extracts and/or, (b) the interaction between the antigen (AFB1) and the antibody is hindered in an immunoassay (
The principle of aflatoxin quantification is similar to cELISA. However, an enzyme label, radio isotope-labeled antibodies or aflatoxin standards are used as reporter molecules. The specific activity of the radioactive marker plays an important role in determining the sensitivity of the assay. Although 14C, 3H, and 125I labeled mycotoxins have been used in various RIAs, 3H labeled toxins were most commonly used. However, 125I labeled toxins have been shown to provide the highest sensitivity (
The RIAs have been used for analysis of aflatoxins in corn, wheat, peanut, milk, serum and eggs with a minimum detection limit ranging from 0.25–0.5 ng in each assay when titrated mycotoxins are used as the marker. However, because of sample matrix interference, the lower limit of mycotoxin detection in food or feed samples is about 2–5 µg/kg. The sensitivity of the RIA can be improved by a sample clean-up procedure after extraction. Although RIA provides sensitive and accurate mycotoxin analysis, use of radioactive legend poses difficulties in safe disposal. Thus, it has been primarily used in the laboratories permitted to use radioisotopes. As a consequence, it mainly remained as a research tool in well established laboratories.
By shortening the incubation time and adjusting antibody and toxin-enzyme conjugate concentrations in the direct or modified indirect competitive ELISA system, it is possible to do a quick screening test at certain toxin levels. Based on the principle of antigen-antibody interactions such as in ELISA, several other immuno-screening tests with sensitivity similar to ELISA have been developed. In this, the antibody is immobilized on a paper disk or other affinity membranes, which is used directly as a strip or mounted either on a plastic card or a cup. The reaction is carried out on the wetted membrane disk. Upon completion, the absence of color (or decrease in color that is generally blue) at the sample spot indicates the presence of toxin in the sample; and the entire test can be completed in less than 1 hr.
Another test is the immunoaffinity method, which is applicable to mycotoxins such as aflatoxins that have fluorescence. In this assay, aflatoxin extracted from the sample is first diluted with buffer at pH 7.0 and subjected to disposable affinity column containing anti-aflatoxin antibody coupled with Sepharose gel. Samples such as milk and urine can be applied to the column directly after adjusting the pH and dilution. After washing, aflatoxin is removed from the column with the methanol, subjected to treatment with iodine/bromine solution, and fluorescence is determined. The affinity column serves as a specific clean-up and concentration tool for further analysis by HPLC method (
The cELISA tests has provided a unique opportunity for ICRISAT and its partners to select breeding populations possessing resistance to aflatoxin contamination, and to evaluate food, feed and related commodities for aflatoxin contamination. This is contributing to stimulate appropriate interventions to enhance the food and human health safety and enhance trade and farmers' incomes. ICRISAT helped in establishing 17 aflatoxin monitoring laboratories in India, Mozambique, Kenya, Malawi and Mali that use our cELISA technologies. Training was provided to the local personnel to manage the facilities. The diagnostic reagents are widely distributed to partners in Asia and Sub-Sahara Africa. These laboratories are contributing to the quality certification of the farmers produce and enhancing the competitiveness of the produce in domestic and international markets. For instance, National Small Farmer Association of Malawi (NASFAM) and ICRISAT have established collaboration for testing the peanut produced for aflatoxin content. Based on the level of contamination, NASFAM graded peanut lots into permissible (<4 µg kg−1 or <20 µg kg−1) and non-permissible (>20 µg kg−1). Graded peanut lots found favorable market for regional and global export, benefiting the farmers (
1 Principal Scientist and Director, West and Central Africa, ICRISAT, BP 12404, Niamey, Niger.
2 Scientific Officer, ICRISAT, Patancheru 502 324, Andhra Pradesh, India.
3 Virologist, IITA, Oyo Road, PMB 5320, Ibadan, Nigeria.