Overview - EN ISO 14675:2003 (Milk and milk products / Aflatoxin M1)
EN ISO 14675:2003 (ISO 14675 / IDF 186) provides guidelines for a standardized description and performance expectations of competitive enzyme immunoassays (EIAs/ELISAs) used to screen for aflatoxin M1 (AFM1) in milk and milk products. The standard is intended to harmonize how quantitative competitive enzyme immunoassays are described, evaluated and reported, and clarifies that positive screening results require confirmation by an accepted reference method for legal purposes.
Key topics and technical requirements
The standard specifies practical assay parameters and statistical expectations for competitive ELISA formats (commonly 96‑well microtitre plate assays):
- Assay principle: Competitive enzyme immunoassay (direct or indirect competitive formats).
- Reagents: Polyclonal or monoclonal antibodies; labelled antigen using horseradish peroxidase (e.g., aflatoxin B- or M‑oxime–HRP).
- Assay format & time: 96‑well microtitre plate; typical duration 3–4 hours.
- Sensitivity / analytical range: Recommended standard curve concentration range ~5 ng/L to 50 ng/L (or broader); example sensitivity targets in the standard (e.g., concentrations giving 80% and 50% relative absorbance).
- Performance metrics:
- Standards: CV of repeatability (relative absorbance) < 10%; CV of reproducibility < 20%.
- Samples (example values): repeatability limit < 100 ng/kg at 200 ng/kg (milk powder); reproducibility limit < 150 ng/kg at 200 ng/kg.
- LOD < 5 ng/kg (milk); LOQ < 10 ng/kg (milk).
- Recovery > 80% for 10–50 ng/kg (milk).
- Statistical/curve fitting: Use of cubic spline, four-parameter logistic models or linear regression (for linear portion of curve); at least six standard dilutions (including zero) and two or more replicates.
- Sample prep: Defatting by centrifugation; reconstitution of milk powder as appropriate.
Applications and users
EN ISO 14675:2003 is intended for:
- Dairy industry quality control and routine screening to document absence of AFM1 above regulatory limits.
- Food safety laboratories and contract testing facilities that implement competitive EIAs/ELISAs for rapid screening.
- Regulators and inspection bodies that specify screening workflows and confirmatory testing pathways.
- Test kit manufacturers and method developers for standardized method descriptions, validation and claims.
Practical uses include on‑site or analytical-lab screening of raw milk, processed milk and milk powder, batch release checks, monitoring programs and surveillance for compliance with aflatoxin M1 regulatory limits.
Related standards and context
- Published jointly by ISO and IDF (and developed with AOAC collaboration), this guideline complements national and international reference/confirmatory methods (used to validate positive EIA results). Use EN ISO 14675 alongside mandated confirmatory analytical procedures when enforcement action or legal evidence is required.
Keywords: EN ISO 14675:2003, aflatoxin M1, competitive enzyme immunoassay, ELISA, milk and milk products, screening methods, limit of detection, assay sensitivity, 96‑well microtitre plate.