D2.284 - Peanut Materials Used for Immunoassay Calibration Exhibit Distinct Allergen Profiles

A range of allergen source materials are used for the calibration of peanut allergen immunoassays. Previous studies have demonstrated that the use of different calibration source materials can result in differences in allergen quantification.

The aim of this study was to further investigate differences between calibration sources by assessing the relative profiles of the major peanut allergens Ara h 2, Ara h 3, and Ara h 6, normalised to total protein content, in two materials recommended for food allergen immunoassay calibration. 

NIST SRM 2387 peanut butter (NPB) and roasted peanut flour (RPF) were selected for comparison as recommended materials by AOAC International. Total protein content of NPB and RPF was determined by Kjeldahl analysis. Protein content was calculated from nitrogen using a conversion factor of 5.46. Twelve extracts of each material were prepared using an optimised extraction protocol. Extracts were analysed for specific allergen content using allergen-specific immunoassays across four plates and two operators. Specific allergen content was expressed as a proportion of total protein.

Extracted specific allergen content in peanut flour and peanut butter varied considerably even when corrected for material protein content. Extracted peanut allergens Ara h 2, Ara h 3 and Ara h 6 represented 16.48 (±12%) µg/mg, 41.41 (±9%) µg/mg and 9.22 (±12%) µg/mg of total protein respectively in RPF. In NPB, specific allergen levels represented 10.61 (±7%) µg/mg, 119.84 (±12%) µg/mg and 5.25 (±8%) µg/mg total protein. Ara h 2 proportion of total protein was most consistent between sources (1.6-fold difference) and Ara h 3 proportion of total protein presented greatest variation between sources (3-fold difference).

These findings demonstrate the specific allergen profile can vary between food allergen materials used for immunoassay calibration and is not directly linked to total protein content. These data provide explanation for differences in assay quantification observed when different calibration sources are used.

These data may also be used as conversion factors from specific allergen content to total food commodity protein for immunoassays reporting in specific allergen content for a given food source (e.g. light roast peanut flour).