| AOAC GFA Program (Food Allergens) | 1 |
| Contents | 2 |
| Guidance on Food Allergen Immunoassay Validation | 4 |
| 1. Scope | 4 |
| 2. Applicability | 4 |
| 3. Terms and Definitions | 4 |
| 3.1 General | 4 |
| 3.1.1. Analyte | 4 |
| 3.1.2. Enzyme-linked Immunoassay (ELISA) | 4 |
| 3.1.3. Food Allergen | 4 |
| 3.1.4. Food Allergen Material | 4 |
| 3.1.5. Matrix | 4 |
| 3.1.6. Measurand | 4 |
| 3.1.7. Test Material | 4 |
| 3.1.8. Spiked Test Material | 4 |
| 3.1.9. Incurred Test Materials | 4 |
| 3.1.10. Qualitative Method | 4 |
| 3.1.11. Quantative Method | 5 |
| 3.1.12. Reference Material | 5 |
| 3.1.13. Robustness | 5 |
| 3.1.14. Sample | 5 |
| 3.1.15. Selectivity | 5 |
| 3.1.16. Technical Replicate | 5 |
| 3.2 Quantitative Methods | 5 |
| 3.2.1. Bias | 5 |
| 3.2.2. Calibrant | 5 |
| 3.2.3. Limit of Detection (LOD) | 5 |
| 3.2.4. Limit of Quantification (LOQ) | 5 |
| 3.2.5. LOQ RSD | 6 |
| 3.2.6. Measurement Range | 6 |
| 3.2.7. Recovery | 6 |
| 3.2.8. Precision | 6 |
| 3.2.9. Repeatability | 6 |
| 3.2.10. Reproducibility | 6 |
| 3.2.11. Intermediate Precision | 6 |
| 3.3 Qualitative Methods | 6 |
| 3.3.1. Probability of Detection (POD) | 6 |
| 3.3.2. Claimed Detection Capability (CDC) | 6 |
| 3.3.3. Fractional Recovery | 6 |
| 3.3.4. Lateral Flow Device (LFD) | 6 |
| 3.3.5. LPOD | 6 |
| 3.3.6. Sensitivity | 7 |
| 4. Required Method Information | 7 |
| 4.1 General Method Information | 7 |
| 4.1.1. Scope | 7 |
| 4.1.2. Applicability Statement | 7 |
| 4.1.3. Standard Method Performance Requirements (SMPR) | 7 |
| 4.1.4. Analyte and Measurand | 7 |
| 4.2 Reporting Units | 8 |
| 4.2.1. At a minimum for food... | 8 |
| 4.2.2. Method developers may provide... | 8 |
| 4.2.3. For environmental surface samples... | 8 |
| 4.3 Calibrant | 8 |
| 4.3.1. What is the calibrant for the method? | 8 |
| 4.3.2. How was the concentration value of the calibrant assigned? | 8 |
| 4.3.3. Is the calibrant made from raw or processed material? | 8 |
| 4.3.4. Was the calibrant extracted or purified? If so, how? | 8 |
| 4.3.5. How is the concentration of the calibrant expressed? | 8 |
| 4.3.6. Is the calibrant commercially available? | 8 |
| 4.4 Testing Material Preparation | 8 |
| 4.4.1. Method developers must provide complete information... | 9 |
| 4.4.2. Specific guidance on allergen materials and test material preparation can be found in Annex A. | 9 |
| 4.5. Antibody Description | 9 |
| 4.5.1. Method developers must provide information... | 9 |
| 4.5.2. Information on the antibody must include the following: | 9 |
| 5. Quantitative Method Validation | 9 |
| 5.1. General Study Practices | 2 |
| 5.1.1. Method developers may prepare study test materials... | 9 |
| 5.1.2. Ideally, all test materials for the independent laboratory a | 9 |
| 5.2. Single Laboratory Validation Study (Method Developer Study) | 9 |
| 5.2.1. Scope | 9 |
| 5.2.2. Calibration Fit Study | 9 |
| 5.2.3. Selectivity Study | 10 |
| 5.2.4. Matrix Study | 2 |
| 5.2.5. Robustness Study | 2 |
| 5.3. Independent Laboratory Study (PTM) | 2 |
| 5.3.1. Scope | 2 |
| 5.3.2. Matrix Study | 2 |
| 5.4. Collaborative Study | 2 |
| 5.4.1. AOAC Requirements | 2 |
| 5.4.2. Scope | 2 |
| 5.4.3. Number of Laboratories | 2 |
| 5.4.4. Test Materials | 2 |
| 5.4.5. Data Analysis | 2 |
| 5.4.6. Acceptance Criteria | 2 |
| 6. Qualitative Method Validation | 2 |
| 6.1. General Study Practices | 2 |
| 6.2. Scope | 2 |
| 6.2.1. Scope | 2 |
| 6.2.2. Selectivity Study | 2 |
| 6.2.3. Matrix Study | 2 |
| 6.2.4. Robustness Study | 2 |
| 6.2.5. High-Dose Hook Effect Study | 2 |
| 6.3. Independent Laboratory Study (PTM) | 2 |
| 6.3.1. Scope | 2 |
| 6.3.2. Matrix Study | 2 |
| 6.4. Collaboratory Study | 2 |
| 6.4.1. AOAC Requirements | 2 |
| 6.4.2. Scope | 3 |
| 6.4.3. Test Materials | 3 |
| 6.4.4. Study Design | 3 |
| 6.4.5. Data Analysis, Results Reporting, and Acceptance Criteria | 3 |
| 6.4.6. Collaborator Comments | 3 |
| Tables | 8 |
| Table 1: Principal Measurands, Analytes, & Measurement Systems for Allergens | 8 |
| Table 2: Required Compounds for Selectivity Study for All Allergen Methods | 13 |
| Table 3: Required Compounds for Selectivity Testing for Target Allergen Test Methods | 14 |
| Table 4: Optional Compounds of Interest for Selectivity Testing | 15 |
| Table 5: Required Test Materials for Quantitative Study Designs | 17 |
| Figures | 19 |
| Figure 1 | 19 |
| Figure 2 | 19 |
| Figure 3 | 20 |
| Figure 4 | 21 |
| Figure 5 | 21 |
| ANNEX A: Preparation of Food & Ingredient Testing Materials for Food Allergen Method Validation | 36 |
| Annex A - 1. Food Allergen Materials | 36 |
| Annex A - Table A1: Example Food Allergen Materials | 36 |
| Annex A - 2. Matrix Formulation and Processing Parameters | 37 |
| Annex A - 3. Incurred Matrix Preparation | 37 |
| Annex A - 4. Spiked Samples | 39 |
| Annex A - References | 40 |
| ANNEX B: Preparation of Environmental Samples for Food Allergen Method Validation | 41 |
| Annex B - 1. Food Allergen and Surface Sampling Materials | 41 |
| Annex B - 2. Application of Food Allergen Materials to Surfaces | 41 |
| Annex B - 3. Method developer shall validate the surface application relevant to their claim | 41 |
| Annex B - 4. Surface Swab Sample Collection | 42 |
| Annex B - 5. Interference from cleaning and sanitizing/disinfecting agents | 42 |
| Annex B - 6. Storage and shipment of sample | 42 |
| Annex B - 7. Sample preparation. | 42 |
| ANNEX C: Statistical Methods: Data Analysis Guidance & example Datasets | 43 |
| Annex C - 1. Intermediate Precision and Repeatability Estimation from Nested Designs: | 43 |
| Annex C - 1.1. | 43 |
| Annex C - 1.2. Basic Principles of the Nested Designs | 43 |
| Annex C - 1.2.1. Defining the Variance Components: | 43 |
| Annex C - 1.3. What is a “Nested” experiment?... | 44 |
| Annex C - 1.3.1. Nested experiments... | 44 |
| Annex C - 1.3.2. Proposed Decision Rules for Determining Nested Variables | 45 |
| Annex C - 1.4. Model Statements in R | 45 |
| Annex C - 1.4.1. For the nested ANOVA analysis... | 45 |
| Annex C - 1.4.2. Example Code for 3-level ANOVA (Designs 1a and 1b): | 46 |
| Annex C - 1.4.3. Example Code for 4-level ANOVA (Designs 2a and 2b): | 46 |
| Annex C - 1.4.4. Note... | 46 |
| Annex C - 1.5 Example Code with Datasets | 44 |
| Annex C - 1.5.1. Data set A1a for Design 1a: | 47 |
| Annex C - 1.5.2. | 47 |
| Annex C - 1.5.3. R-Code for Data Set A1a | 47 |
| Annex C - 1.5.4. Data Output | 48 |
| Annex C - 1.5.5. Reporting of Precision Estimates | 48 |
| GFA TEST DATA RESULTS PLOT SET A1a | 49 |
| Annex C - 1.5.6. Data Set A2b | 50 |
| Annex C - 1.5.7. R-Code for Data Set A2b | 50 |
| Annex C - 1.5.8. Data Output | 51 |
| Annex C - 1.5.9. Use the same code for Design 2a | 51 |
| Annex C - 1.5.10. Reporting Precision Estimates | 51 |
| GFA TEST DATA RESULTS PLOT SET A2b | 53 |
| Annex C - 2. Limit of Detection and Limit of Quantification Estimation | 53 |
| Annex C - 2.1. Limit of Detection (LOD) | 53 |
| Annex C - 2.1.1. | 53 |
| Annex C - 2.1.2. Calculations: | 53 |
| Annex C - 2.1.3. Multiple Matrices: | 53 |
| Annex C - 2.1.4. Example Data and Calculations | 53 |
| Annex C - Table 1: EXAMPLE DATA TABLE | 54 |
| Annex C - 2.2. Limit of Quantification (LOQ) | 55 |
| Annex C - 2.2.1. Data required: | 55 |
| Annex C - 2.2.2. Model %RSDi | 55 |
| Annex C - 2.2.3. Plot %RSDi vs. mean concentration | 55 |
| Annex C - 2.2.4. Estimate LOQ... | 55 |
| Annex C - 2.2.5. Values used as estimates... | 55 |
| Annex C - 2.2.6. Prepare an operator characteristic (OC) curve for LOQ | 55 |
| Annex C - 2.2.7. Calculations/procedures for multiple matrices | 56 |
| Annex C - 2.2.8. Example Data and Calculations | 56 |
| Probability of a Quantitative Result w/LOQ=0.61 ppm | 57 |