AOAC Guidance on FA Immunoassay Validation (August 2023)

1.6. Method developers must provide the following information for reference and alternative materials: 1113 1.6.1. Total protein content (Dumas or Kjehldahl, N-conversion factor should be stated) 1114 1.6.2. Processing steps to obtain the material if possible/known. 1115 1.6.3. Source of the material (e.g. genetic variety/subspecies/geographic origin) if possible/known. 1116 1.6.4. Commercial supplier of the material, if applicable. 1117 1.6.5. Protein profile data using SDS-PAGE is required to demonstrate comparability between 1118 reference and alternate materials. The method developer should share the following for 1119 reference and alternate materials: protein profile data, protein's size and description, and 1120 protein molecular weight (e.g., label bands and gel percentage). Western blotting/immunoblot 1121 assay with the appropriate antibody should also be presented and labeled, including the 1122 detection method. Mass spectrometry, HPLC and literature references are optional alternatives. 1123 1.7. When preparing testing materials, unaltered food allergen material (see Table A1) should be used 1124 instead of a protein extract of the food allergen material. 1125 2. Matrix Formulation and Processing Parameters 1126 2.1. The formulation and processing of incurred matrices should be representative of typical recipes 1127 used for standard consumption. 1128 2.1.1. Kitchen or restaurant matrix formulations and processing conditions are largely appropriate. 1129 2.1.2. In some cases, independent organizations may have standardized protocols for relevant 1130 matrix preparation (e.g., Cereals and Grains Association-AACC, CODEX). 1131 2.2. The incurred material recipe and preparation should be detailed in the final report. 1132 2.2.1. Follow CODEX STAN 87-1981 for minimum requirements in chocolate. 1133 2.3. In the case of a claimed matrix that has been minimally processed (e.g., ingredients such as wheat 1134 flour, corn flour, spice blends, etc.) appropriate testing material preparation most likely includes 1135 thorough mixing of the reference material or other food allergen material with a commercial 1136 source of the matrix, without additional processing. 1137 3. Incurred Matrix Preparation 1138 3.1. It is advisable to pre-screen all ingredients for the presence of undeclared target food allergen. 1139 3.2. Ideally, each ingredient used for incurred matrix preparation should come from the same 1140 production lot (i.e., have the same lot codes). 1141 3.3. All equipment used for incurred matrix preparation must be clean and/or dedicated equipment 1142 should be used for blank and incurred matrix preparations. Preparation of blank matrices, followed 1143 by increasing levels (low to high incurred levels) may be advisable to prevent impacts of any cross- 1144 contact between batches. 1145 3.4. For most food matrices, test-kitchen-scale or restaurant-scale equipment will be suitable for 1146 incurred matrix preparation, such as baked and heated products. Foods that are processed with 1147 operations requiring pilot or industry scale equipment (e.g. extrusion, high-pressure processing, 1148 fractionation, hydrolysis, etc.), may be prepared by a qualified external food processing entity. 1149 3.5. For incurred matrices, the food allergen material is generally first blended homogeneously with one 1150 or more ingredients with similar properties (see also 3.8.1. and 3.8.2.), followed by incorporation of 1151