AOAC Guidance on FA Immunoassay Validation (August 2023)

Annex B

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Preparation of Environmental Samples for Food Allergen Method Validation

1. Food Allergen and Surface Sampling Materials 1254 1.1. For information on the selection and characterization of food allergen materials, see Annex A. 1255 1.2. All information for food allergen materials described in Annex A is also required to be reported for 1256 materials used to prepare environmental surface samples. 1257 1.3. For environmental surface samples, method developers must also describe the swabbing/sponge 1258 material used for the validation and disclose any known issues with surface compatibility. 1259 2. Application of Food Allergen Materials to Surfaces 1260 2.1. Create a high-level concentration of the food allergen material (200–1,000 mg total protein/kg 1261 suspension) in a dispersant diluent suitable for the matrix (e.g., phosphate buffered saline). 1262 2.1.1. Prior to preparation of the suspension, the food allergen material should be comminuted to a 1263 particle size that is practical for assuring homogeneity in the diluent. 1264 2.1.2. Once prepared, this suspension should be used within 24 hours. To use a suspension after 24 1265 hours, the stability of the suspension must be demonstrated for relevant time periods. 1266 2.1.3. It is advisable for method developers to understand the homogeneity of the materials used to 1267 contaminate surfaces. Use of a test material with heterogeneous food allergen material 1268 distribution will deliver higher than expected levels of variance in method performance. 1269 Homogeneity testing may be performed by analyzing 10 test portions of the food allergen 1270 suspension with an appropriate quantitative ELISA method. 1271 2.2. Adjust the stock spike suspension to achieve the indicated levels. Inoculate each surface test area 1272 with a known volume that will allow for even distribution over the entire test surface area without 1273 producing excessive accumulation of liquid. Use blank spiking solution for non-contaminated surface 1274 areas. 1275 2.2.1. Example of level to apply: 250 µL of 4 µg/mL equal to 1 µg allergen protein applied evenly 1276 over the defined area (e.g., 10x10 cm 2 ). 1277 2.2.2. Approved food contact material surfaces that should be considered environmental matrices 1278 are those that comply with local requirements (e.g., U.S. FDA 21 CFR parts 110 173, 177, 1279 180.22, 181). 1280 2.2.3. Typically, surface testing validation includes stainless steel, plastic, cast iron, ceramic, and 1281 rubber materials. Also types of items such as conveyor belt links, baking pans and spatulas 1282 which may be made of these materials should be considered in the validation. 1283 3. Method developer shall validate the surface application relevant to their claim. 1284 3.1. For dry surface testing, allow inoculated surfaces to dry for 16-24 h at room temperature (20-24°C). 1285 Surfaces must be visibly dry before sampling. 1286 3.1.1. Caution: Consider location for validation to prevent cross contamination and ensure the 1287 surface are visibly clean. 1288 3.2. For wet surface testing, test within 30mins-1 h of inoculation. Surfaces should be visibly wet before 1289 sampling. 1290