BS EN ISO 14675:2003 – Milk and milk products — Guidelines for a standardized description of competitive enzyme immunoassays — Determination of aflatoxin M 1 content
1 Scope
This International Standard give guidelines on the use of screening methods used for the determination of aflatoxin M 1 content in milk and milk products, based upon competitive enzyme immunoassays.
For legal purposes, positive enzyme immunoassay results require confirmation by an accepted reference method.
However, depending on whether the test complies with the specifications given hereafter, enzyme immunoassays can be used for routine quality control, especially when the absence of aflatoxin M 1 above the regulatory limit needs to be documented.
2 Principle
Immunochemical methods are based on the ability of antibodies to bind to specific substances. The reversible
association between antibodies and their corresponding antigens is called the immunological reaction. The binding
forces involved are weak molecular interactions, such as Coulomb and van der Waals forces, as well as hydrogen bonds and hydrophobic binding.
The antigen-antibody reaction is based on the law of mass action and the amount of antigen or antibody present in the reaction mixture can be inferred from the extent of the reaction.
The “quality” of any immunoassay is a function of the immunochemical principle of the method, the properties of the reagents, the assay design and the experimental errors. These basic principles determine the sensitivity,specificity, precision and accuracy of the assay.
Concerning the principle of the method, a distinction exists between competitive methods and noncompetitive methods.
For practical reasons, these methods need either labelled antigen or labelled antibody to permit observation of the antigen-antibody reaction.
Competitive methods are based on the competition of free (A ) and labelled (A *) antigen for a limited number of g g antibody-combining sites (AB).
Schematically, this immunochemical principle may be presented according to the following formula:
In most cases, the assay response represents the bound-labelled antigen, but any measure of the distribution of the labelled antigen is, in principle, possible. For the detection of low molecular weight compounds like mycotoxins, which possess only one antibody binding site (epitope), the competitive assay format is mandatory.
To provide a distinction between unreacted and complexed reactants, most assays use either antibody (direct competitive assay) or antigen (indirect competitive assay) bound to a solid-phase as immunosorbent. So all the reagents that are not bound by the antibody can be easily removed by “washing” the solid phase.
BS EN ISO 14675:2003 – Milk and milk products — Guidelines for a standardized description of competitive enzyme immunoassays — Determination of aflatoxin M 1 content