BS EN ISO 14675 pdf download

BS EN ISO 14675 pdf download Milk and milk products — Guidelines for a standardized description of competitive enzyme immunoassays — Determination of aflatoxin M 1 content
Scope
This lnternational Standard give guidelines on the use of screening methods used for the determination of aflatoxinMi content in milk and milk products, based upon competitive enzyme immunoassays.
For legal purposes, positive enzyme immunoassay results require confirmation by an accepted reference methodHowever, depending on whether the test complies with the specifications given hereafter, enzyme immunoassayscan be used for routine quality control, especially when the absence of aflatoxin M1 above the requlatory limitneeds to be documented.
2 Principle
lmmunochemical methods are based on the ability of antibodies to bind to specific substances. Thereversible ssociation 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 thesensitivity, specificity, precision and accuracy of the assay.
Concerning the principle of the method, a distinction exists between competitive methods and non.competitive methods.
For practical reasons, these methods need either labelled antigen or labelled antibody to permit observation ofthe antigen-antibody reaction.
Competitive methods are based on the competition of free (A ) and labelled (A *) antigen for a limited number of 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.
3 Aflatoxin M 1 enzyme immunoassay
Based on the information given in the general principles of immunoassays (see clause 2), it is recommended that an ELISA-method for aflatoxin M 1 should comply with the specifications given in Table 1 .