Enzyme immunoassay for the quantitative determination of Aflatoxin M1 in milk and milk products.
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.
Aflatoxin M1 was the first metabolite of Aflatoxin B1, which could unequivocally be detected by Allcroft and Carnaghan in the milk of cows in 1963. Out of this reason this first derivative was called Aflatoxin M1 (= milk). As further investigations showed, also other mammalians excrete Aflatoxin M1 in milk, feces and urine. Contaminations of milk and milk products can be hazardous for human beings, because M1 is similar to Aflatoxin B1 regarding its hepatotoxicity. M1 is only less carcinogenic.
In order to protect people against aflatoxin-induced diseases, there is a need for the qualitative and quantitative control of endangered foodstuff, besides appropriate hygienic precautions, which avoid the formation of aflatoxins. The Aflatoxin M1 ELISA is a quick, economical and sensitive method to detect aflatoxin M1 in milk and milk products. After an appropriate sample preparation, 40 samples can be tested in duplicate within 140 minutes.
Description: The Aflatoxin M1 quantitative test is based on the principle of the enzyme linked immunosorbent assay. An aflatoxin conjugate is bound on the surface of a microtiter plate. Aflatoxin M1 containing samples or standards and an antibody directed against aflatoxin M1 are given into the wells of the microtiter plate. Immobilized and free aflatoxin M1 compete for the antibody binding sites. After one hour incubation at room temperature, the wells are washed with diluted washing solution to remove unbound material. A peroxidase conjugate against the antibody is given into the wells and after another hour incubation, the plate is washed again. Then a substrate solution is added and incubated for 20 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 aflatoxin M1 is indirectly 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 M1 Standards: 0, 100, 500, 1000, 5000, 10000 pg/mL;
- ELISA reader at 450 nm;
- Standard range: 10 – 1000 pg/mL;
- Sensitivity analytical: <10 pg/mL.