ISO 14675:2003

Overview

ISO 14675:2003 / IDF 186:2003 provides guidelines for a standardized description and basic validation parameters of competitive enzyme immunoassays (EIAs/ELISAs) used to determine aflatoxin M1 in milk and milk products. The standard focuses on screening methods - primarily the common 96‑well microtitre plate competitive ELISA format - and explains requirements for sensitivity, specificity, precision, curve fitting and sample preparation. It emphasizes that positive EIA results must be confirmed by an accepted reference method for legal or regulatory purposes, while validated EIAs can be used for routine quality control and documentation that aflatoxin M1 levels are below regulatory limits.

Key Topics and Technical Requirements

• Assay format and principle
  • Competitive enzyme immunoassay (96‑well microtitre plate); typical assay time 3–4 hours.
• Reagents
  • Antibody: polyclonal or monoclonal.
  • Labelled antigen: horseradish peroxidase (e.g., aflatoxin B- or M‑oxime–HRP conjugate).
• Sensitivity and specificity
  • Target assay sensitivity: relative binding values around 80% at <20 ng/L and 50% at <50 ng/L.
  • Cross‑reactivity: aflatoxin M1 = 100%; other aflatoxins occurring in milk < 20%.
• Statistical design and curve fitting
  • Standards: ≥2 replicates, ≥6 dilutions including zero; concentration range typically 5–50 ng/L or broader.
  • Curve fitting: four‑parameter logistic or cubic spline recommended (linear regression only on linear region).
• Precision and limits
  • CV of repeatability (relative absorbance) <10%; reproducibility <20%.
  • Sample performance targets: LOD <5 ng/kg (milk), LOQ <10 ng/kg (milk).
  • Repeatability/reproducibility at the 200 ng/kg milk‑powder level: <100 ng/kg and <150 ng/kg respectively.
• Sample preparation and recovery
  • Defat by centrifugation; reconstitute milk powder to milk; recovery >80% for 10–50 ng/kg range.
• Limitations
  • Matrix effects and cross‑reactivity can cause false positives; confirmation by reference methods is required for legal action.

Applications and Who Uses It

• Routine quality control laboratories in the dairy industry documenting compliance with maximum limits for aflatoxin M1.
• Food safety and regulatory laboratories implementing screening before confirmatory testing.
• Test kit manufacturers and method developers standardizing performance claims for competitive ELISAs.
• Third‑party auditors and certification bodies assessing laboratory screening procedures.

Related Standards and References

• Published jointly by ISO and IDF (prepared with AOAC collaboration). For regulatory enforcement, EIA screening results should be confirmed by an accepted reference method (national or international reference methods/AOAC standards). For method validation and curve‑fitting details see the statistical references cited in ISO 14675:2003.

Keywords: ISO 14675, aflatoxin M1, ELISA, enzyme immunoassay, milk testing, competitive immunoassay, aflatoxin screening, 96‑well microtitre plate, limit of detection, method validation.