Enzyme immunoassay for the quantitative determination of Aflatoxins in food.
Background: Aflatoxins belong to the class of mycotoxins. Chemically they are defined as difuranocyclopentanocumarines or difuranopentanolidocumarines, i.e. aflatoxins contain a dihydrofuran or a tetrahydrofuran ring, to which a substituted cumarin system is condensed. Out of about 20 known aflatoxins, the moulds Aspergillus flavus and A. parasiticus produce exclusively aflatoxin B1, B2, G1 and G2, and all the other aflatoxins are derivates of these four. The derivates are developed either by metabolism in humans, animals and microorganisms or by environmental reactions. In the European Union the limits for total aflatoxins are 4 – 15 ppb for food. Thus a monitoring of food and feed with respect to the concentration of aflatoxins is obligatory.
Aflatoxin total ELISA represents a highly sensitive detection system and is particulary capable of the quantification of total aflatoxin contaminations in cereals, beer, nuts, dry fruits, oils and chili.
Description: The Aflatoxin total quantitative test is based on the principle of the enzyme-linked immunosorbent assay. An antibody binding protein is coated on the surface of a microtiter plate. Aflatoxins containing samples or standards, an aflatoxin-peroxidase conjugate and an antibody directed against aflatoxins are given into the wells of the microtiter plate. The conjugate competes with the aflatoxins of samples/standards for the limited number of antibody sites. Simultaneously the anti-aflatoxin antibody is bound to the antibody-binding protein coated on the microtiter plate. After 30 minutes incubation at room temperature the wells are washed with diluted washing solution to remove unbound material. A substrate solution is added and incubated for 15 minutes, resulting in the development of a blue colour. The colour development is inhibited by the addition of a stop solution, and the colour turns yellow. The yellow colour is measured photometrically at 450 nm. The concentration of aflatoxins is inversely proportional to the colour intensity of the test sample.
- The kit contains reagents for 96 determinations;
- Microtiter plate consisting of 12 strips with 8 breakable wells;
- Aflatoxin Standards: 0, 0.05, 0.1, 0.25, 0.5, 1.5 ng/mL;
- ELISA reader at 450 nm;
- Standard range: 0.05 – 1.5 ng/mL;
- Sensitivity analytical: 0.015 ng/mL.