Standard Materials for Allergen Analysis and Their Use in a Ring Trial Validation

A new multiplex real-time PCR method for detecting brown/black mustard, white mustard, celery and pistachio was validated in an 11-laboratory ring trial, using specially produced standard allergen mixtures incurred into five foods.

The results of the interlaboratory validation of a new multiplex real-time PCR method for the detection and semi-quantitative determination of the allergenic species brown/black mustard, white mustard, celery, and pistachio in food are presented in this article. A new approach was pursued, based on specially produced standard allergen stock mixtures. These pre-mixtures were incorporated (incurred) into five different foods on a laboratory scale and further processed (sausage, cookies, chocolate, ice cream, falafel).

The results show that the allergen mixtures are well suited for the production of calibration standards for allergen analysis on the one hand and for the production of specific matrix control samples on the other. With the help of these materials, the multiplex PCR method was successfully validated in a ring trial involving 11 laboratories. Detection of the allergenic ingredients was possible in the range of 1.0 mg/kg (i.e., approx. 0.2–0.3 mg allergenic proteins/kg).

The suitability for – at least – semi-quantitative estimation of the allergenic ingredients was confirmed by analysing the five matrix control samples spiked in the range of 10–50 mg/kg. The new approach allows the flexible verification of the suitability of the method for the matrix relevant to control practice. In the interlaboratory test, sufficient recovery rates and measurement uncertainties were determined for three matrices. Too low recoveries were obtained for two matrices (ice cream, falafel), which led to measurement uncertainties of more than 50%.

Keywords

• Allergens
• Standard materials
• Multiplex Real-time PCR
• Inter-laboratory validation
• Quantification