Afla-ELISA: A test for the estimation of aflatoxins

Lava Kumar (L.kumar@cgiar.org) and R. Bandyopadhyay
L. Kumar, IITA’s Head of Germplasm Health Unit and Virologist; R. Bandyopadhyay, Plant Pathologist, IITA, Ibadan, Nigeria

Aflatoxin testing using Afla-ELISA. Source: L. Kumar.
Aflatoxin testing using Afla-ELISA. Source: L. Kumar.
Aflatoxins threaten human and animal health
Aflatoxins are the hepatotoxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. prasiticus. They are common contaminants in several staple crops, such as maize and groundnut, produced in the tropics and subtropics. Aflatoxins are a group of four toxins: aflatoxin B1 (AFB1), AFB2, AFG1, and AFG2. A metabolite of aflatoxins, namely AFM1, is detected in milk. Aflatoxin contamination in foods is considered to be unavoidable, as the causative fungi are ubiquitous in the tropical parts of the world. However, fungal infestation and toxin contamination are unpredictable and depend on certain environmental conditions. Aflatoxin exposure in humans and animals results from the consumption of aflatoxin-contaminated foods and feeds.

Regulations check aflatoxin contamination
Stringent food safety regulations are enforced in most countries to prevent use of aflatoxin-contaminated foods and feeds. These programs are executed through a monitoring process by testing all commodities for aflatoxins and rejection of those with toxin levels exceeding the tolerable limits [ranges between 2–20 parts per billion (ppb), depending on the type of toxin and country1]. Heavy infestation of fungi results in moldy products which can be physically sorted. However, aflatoxins per se are invisible and leave no visual clues of their presence in the contaminated products. Aflatoxins can be found even in commodities that show no apparent signs of fungal infestation. This situation poses a serious challenge to monitoring aflatoxin contamination, which depends on aflatoxin-monitoring tools.

Outline of Afla-ELISA testing scheme. Source: L Kumar.
Outline of Afla-ELISA testing scheme. Source: L Kumar.
Aflatoxin control relies on monitoring tools
Monitoring for aflatoxins has become integral to effective measures to control aflatoxins in foods and feeds. A variety of aflatoxin monitoring tools are available to detect and quantify aflatoxin levels2. Quantitative estimation is most critical as decisions are based on aflatoxin levels in the commodity. Products with aflatoxin levels within the permissible range are allowed in trade and those with exceeding levels are rejected1.

Despite the availability of a wide variety of diagnostic tools for monitoring aflatoxins, their use in most of the developing countries is limited by high cost, difficulties with importation, and lack of appropriate laboratory facilities and well-trained staff. Among the many types of aflatoxin-monitoring tools, antibody-based methods were proven to be relatively easy for developing countries to adopt.

Convenient option
At IITA, we developed an enzyme-linked immunosorbent assay (ELISA) named Afla-ELISA, for quantitative estimation of aflatoxins. Very high titered rabbit polyclonal antibodies for AFB1 were produced. These antibodies have an end-point titer of 1:512,000 (v/v) against 100 ng/mL AFB1-BSA standard; they are highly specific to AFB1 and also react with ABF2, AFG1, and AFG2. They were used to develop Afla-ELISA based on the principle of indirect competitive ELISA for quantitative estimation of aflatoxins. This assay has a lowest detection limit of 0.09 ng/mL, and a recovery of 98±10% in maize.

Prototype Afla-ELISA kit―a quantitative serological assay for the estimation of total aflatoxins in maize and other commodities, using 96-well microtiter plates. Up to 20 samples can be tested in each 96-well plate at a cost of US$4 per sample. Source: L Kumar.
Prototype Afla-ELISA kit―a quantitative serological assay for the estimation of total aflatoxins in maize and other commodities, using 96-well microtiter plates. Up to 20 samples can be tested in each 96-well plate at a cost of US$4 per sample. Source: L Kumar.
Afla-ELISA is simple to perform, offers sensitive detection, and is convenient for adoption in sub-Saharan Africa. This test is suitable for routine aflatoxin surveillance in crops and commodities, and offers a low-cost alternative to official monitoring methods. This test offers a sustainable solution to the problem of ever-increasing demand for monitoring programs related to food safety and trade, and has the potential to enhance aflatoxin monitoring capacity in sub-Saharan Africa. To contribute to capacity development, training workshops have been organized on monitoring for mycotoxins and application of Afla-ELISA.

References
1 FAO. 2003. Worldwide regulations for mycotoxins in food and feed. FAO Food and Nutrition Paper #81. FAO, Rome, Italy.
2 Reiter, E. et al. 2009. Mol. Nutr. Food Res. 53: 508–524.

Initiative tackles killer aflatoxin

IITA and partners recently launched a project that will provide farmers in Nigeria and Kenya with a natural, safe, and cost-effective solution to prevent the contamination of maize and groundnut by a cancer-causing poison, aflatoxin. It is funded by the Bill & Melinda Gates Foundation.

Maize cobs attacked by fungi. Photo by IITA.
Maize cobs attacked by fungi. Photo by IITA.

Aflatoxin is produced by a fungus (Aspergillus flavus). It damages human health and is a barrier to trade and economic growth. The toxin, however, is not produced in all strains of the fungus. The project’s biocontrol technology introduces nontoxic strains of the fungus in the affected fields. These “good guys” overpower and reduce the “bad guys,” the population of toxic strains, drastically reducing the rate of contamination.

During the launching of the project, Wilson Songa, Agricultural Secretary in Kenya’s Ministry of Agriculture, said that Kenya welcomed the initiative after recent losses of lives and millions of tons of maize to aflatoxin contamination.

“Kenya has become a hotspot of aflatoxin contamination. Since 2004, nearly 150 people have died after eating contaminated maize,” he said.

IITA had worked with the United States Department of Agriculture to develop a biocontrol solution for aflatoxin, testing it in many fields in Nigeria. The project will take the biocontrol product, commercialize it, and make it available to farmers.

Ranajit Bandyopadhyay, IITA’s plant pathologist, says the project is adding value to previous investments in biocontrol. It will support the final stage of commercialization of aflasafe™ in Nigeria and selection of the most effective strains, development of a biocontrol product, and gathering of data on efficacy in Kenya.

The Nigerian government has joined forces with IITA and the World Bank to help contain the contamination of food crops by aflatoxins.

The collaboration will make aflasafeâ„¢ available to farmers to greatly reduce the aflatoxin menace.

The new approach is part of the Commercial Agriculture Development Program supported by the World Bank and implemented in Kano, Kaduna, Enugu, Cross River, and Lagos States in Nigeria.

In Nigeria, produce from resource-poor maize farmers faces rejection from the premium food market because of aflatoxin contamination.

In on-farm research trials in Kaduna State—north-central Nigeria—during 2009 and 2010, farmers who treated their fields with aflasafe™ were able to reduce the levels of contamination by 80 to 90%.

Related website

Aflatoxin management website – www.aflasafe.com

Investing in aflasafeTM

aflasafeTM is a cost-effective, safe, and natural method for preventing the formation of aflatoxin in maize and other susceptible commodities in the field and also in postharvest storage and processing. It is providing hope for African farmers and opening doors for entrepreneurs looking to invest on a winning formula in the agricultural sector.

Maize farmers receive aflasafeâ„¢ from IITA. Photo by IITA.
Maize farmers receive aflasafeâ„¢ from IITA. Photo by IITA.

Scientific studies suggest that investment in aflasafeTM in Africa is viable, not only for profit but also to improve people’s health. For instance, the study of Wu and Khlangwiset (2010) estimated that the cost-effectiveness ratio (CER; gross domestic product multiplied by disability-adjusted life years saved per unit cost) for aflatoxin biocontrol in Nigerian maize ranged from 5.10 to 24.8. According to the guidelines from the World Health Organization (WHO 2001), any intervention with a CER >1 is considered to be “very cost-effective”.

About aflatoxins
Produced by the fungi Aspergillus spp., aflatoxins are highly toxic fungal substances that suppress the immune system, and cause growth retardation, liver cancer, and even death in humans and domestic animals.

Aflatoxins also affect the rate of recovery from protein malnutrition and Kwashiorkor, and exert severe nutritional interference, including in protein synthesis, the modification of micronutrients, and the uptake of vitamins A and D.

Exposure in animals reduces milk and egg yields. The contamination of milk and meat is passed on to humans after consumption of these products. Aflatoxins affect cereals, oilseeds, spices, tree nuts, milk, meat, and dried fruits. Maize and groundnut are major sources of human exposure because of their higher susceptibility to contamination and frequent consumption.

The toxins are most prevalent within developing countries in tropical regions and the problem is expected to be further exacerbated by climate change.

The high incidence of aflatoxin throughout sub-Saharan Africa aggravates an already food-insecure situation. Agricultural productivity is hampered by contamination, compromising food availability, access, and utilization. Unless aflatoxins in crops and livestock are effectively managed, marketable production and food safety cannot improve. Thus, the economic benefits of increased trade cannot be achieved.
Aflatoxins cost farmers and countries hundreds of millions of dollars annually. These losses have caused crops to be moved out of regions, companies to go bankrupt, and entire agricultural communities to lose stability.

IITA staff producing aflasafeâ„¢ in the lab. Source: R. Bandyophadyay, IITA.
IITA staff producing aflasafeâ„¢ in the lab. Source: R. Bandyophadyay, IITA.

aflasafeâ„¢ to the rescue
An innovative scientific solution in the form of biocontrol has been developed by the US Department of Agriculture’s Agricultural Research Service (USDA-ARS). This breakthrough technology,already widely used in the United States, reduces aflatoxins during both crop development and postharvest storage, and throughout the value chain.

IITA and USDA-ARS have been collaborating since 2003 to adapt the biocontrol for Africa. They achieved significant breakthroughs that resulted in the development of an indigenous aflatoxin technology in Nigeria, now called aflasafeâ„¢. aflasafeâ„¢ contains four native atoxigenic strains of Aspergillus flavus that outcompetes and replaces the toxin-producing strains, thus reducing aflatoxin accumulation.

IITA and partners conducted trials in Nigeria. Native atoxigenic strains reduced contamination by up to 99%. The National Agency for Food and Drugs Administration and Control (NAFDAC) gave IITA provisional registration to begin testing of the inoculum of a mixture of four strains under the trade name aflasafeâ„¢. In 2009 and 2010, maize farmers who applied aflasafeâ„¢ achieved, on average, a reduction of >80% in aflatoxin contamination at harvest and 90% after storage.

Groundnut farmers also achieved more than 90% reduction in Nigeria and Senegal using a version of aflasafeâ„¢ with native atoxigenic strains from Senegal.

In the future
The success recorded so far in the control of aflatoxin comes from aflasafeâ„¢ produced in the lab. Consequently, to meet the demands of farmers in sub-Saharan Africa, large-scale production is needed.

In Nigeria, for instance, nearly 30% of harvested maize has high levels of aflatoxins and is prone to being rejected by the feed industry. In Kenya, last year because of aflatoxin contamination, more than two million bags of maize were declared unfit for human consumption in the Eastern and the Coast provinces. Some countries, such as Senegal, have lost groundnut export market to the European Union due to aflatoxin contamination.

Commercial production of aflasafeâ„¢ would allow easy and widespread availability of a simple solution to the most recalcitrant problem affecting farmers and consumers. The monetized value of lives saved, quality of life gained, and improved trade by reducing aflatoxin far exceeds the cost of aflasafeâ„¢ production.

Reference
Wu F and Khlangwiset P. 2010. Health economic impacts and cost-effectiveness of aflatoxin-reduction strategies in Africa: case studies in biocontrol and post-harvest Interventions. Food Additives & Contaminants. Part A, 27: 4, 496—509, First published on: 05 January 2010 (iFirst).

Related website

Aflatoxin management website – www.aflasafe.com

aflasafeâ„¢: a winning formula

Biological control of aflatoxins using aflasafeâ„¢ is providing hope for African farmers battling with crop contamination and opening doors for the private sector looking to invest on a winning formula in the agricultural sector.

Scientists have developed a cost-effective, safe, and natural method to prevent aflatoxin formation in maize while in the field. Aflatoxin causes liver cancer and suppresses the immune system, endangering both humans and animals. It also retards growth and development of children. This colorless chemical is invisible and its presence and contamination levels can only be confirmed by laboratory tests.

The biocontrol technology works by introducing native (local) strains of the fungus Aspergillus flavus that do not produce the aflatoxin (the ‘good guys’) in the affected fields. This good fungus boxes out and drastically reduces the population of the poison-producing strains (the ‘bad guys’).

The aflasafeâ„¢ technology has the potential to provide relief to millions of maize farmers in sub-Saharan Africa depending on agriculture as a source of livelihood.

According to Ranajit Bandyopadhyay, IITA Plant Pathologist, a single application of this biopesticide 2-3 weeks before maize flowering is sufficient to prevent aflatoxin contamination throughout and beyond a cropping season and even when the grains are in storage.

With an initial investment outlay of US$1−3 million in an aflasafe™ manufacturing plant, investors are likely to reap about $1.33 million annually. Bandyopadhyay said that investing in an aflasafe™ manufacturing plant in Nigeria would pay off considering the huge demand for quality maize in the country. His estimates showed that over 60% of harvested maize in Nigeria currently has high levels of aflatoxins and are prone to being rejected by the feed industry.

Institutions involved in the initiative include IITA, Agriculture Research Service of the United States Department of Agriculture, AATF, and local partners.

Related website

Aflatoxin management website – www.aflasafe.com

O.A. Adenola: More awareness needed on the dangers of aflatoxins

Pastor O.A. Adenola
Pastor O.A. Adenola. Photo by IITA

The president of one of the strongest crop networks in Nigeria, Pastor O.A. Adenola, talks about the need for stakeholders to join forces against aflatoxin spread and other issues. This is an excerpt from his interview with Godwin Atser.

Do farmers understand what aflatoxins are?
They may see the fungus on the maize cob but really many Nigerian farmers do not know the danger in what they see: what it is… what effects it has on people as a result of eating grain that is already contaminated… I think we need a lot of awareness, a lot of teaching to get our farmers to know the dangers of aflatoxins in our foods. The problem is that you don’t see them and their effect physically. If you look at the cassava mealybug, for instance, the farmer sees the plant die. In the case of aflatoxins, you don’t see them causing anything bad to maize; it is the after-effect that damages people’s health.

What can be done to bring the message to the people?
It has to involve a collective effort from all of us: the research institutes, the Agricultural Development Programs, the Maize Association of Nigeria, and the media. We won’t make any progress if we don’t collaborate to get the farmers to know the importance of the effect of aflatoxins on human beings and on animals.

You participated in the Doubling Maize Project. What were your observations?
At the time the project was initiated in 2006, the maize production level on average was 1.5 t/ha. The project target was to double production—from 1.5 to 3 t/ha. A farmer who could not combine production inputs to give us 3 t/ha was not qualified to be involved in the scheme because we did not want to increase the area planted. We wanted to increase production per unit area. The intention was to intensify production so that we could double what was on the ground.

So what happened?
I tell you, farmers made more than 3 t/ha! Also if the technology is properly applied, Nigeria can easily double maize production.

What effort is your association making to disseminate some of the findings of that research to increase maize production?
The maize network is stronger than the networks of other crops in Nigeria, maybe, because of the facilities we have at IITA that are linking us up properly with research and also with Ministries of Agriculture all over the country. And since we were the beneficiaries of the research findings, it was easier for us and for our members to adopt the improved technologies.

All that the researchers were telling us was “You can be better farmers if you take the technology.” I must tell you that every farmer is out there in the field because he wants to make more money. So the benefit is good enough to propel the technology.

How is the collaboration between MAAN and IITA?
Excellent! I have been relating with IITA since 1984 and when this Association was formed in 1992, it was formed in IITA. Since then we have had very good collaboration.

What can IITA do to make this partnership grow?
Whenever there is a need and we call on IITA, they have always answered. The Director General and the maize “chief”, Dr Sam Ajala and his team, have been very cooperative. That collaboration is what is important. If you have a problem and you call your friend and he answers, then you are okay.