AOAC Guidance on FA Immunoassay Validation (August 2023)

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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

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