AOAC Guidance on FA Immunoassay Validation (August 2023)

986 987 988 989 990 991 992 993 994 995 996 997 998 999

results for each sample, as well as the POD and 95% POD confidence interval for each set of 20 replicates. The CDC sample must produce a POD of 0.95 or greater, and the blank sample must produce a POD of 0.05 or less, in each kit. 6.2.4.4.5. Alternatively, method developers may provide internal lot-to-lot and stability data for

review.

6.2.5. High-Dose Hook Effect Study

6.2.5.1. The objective of the hook effect study is to establish an upper working limit, if any.

6.2.5.2. Test Materials

6.2.5.2.1. The test material used for the hook effect study should be the food allergen reference

material or alternative material, as described in Annex A.

6.2.5.3. Study Design

6.2.5.3.1. For each target allergen, perform 5 independent extractions of the source food

allergen material per the kit instructions.

6.2.5.3.2. Dilute each of the 5 extracts 1:10, 1:100, and 1:1000 in the kit extraction buffer. Follow any additional kit instructions to prepare the undiluted and diluted samples

1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011

for analysis.

6.2.5.3.3. Analyze each of the undiluted and diluted samples. 6.2.5.4. Data Analysis, Results Reporting, and Acceptance Criteria

6.2.5.4.1. All results are expected to be positive or specifically indicate overloading. In the event of any negative result, additional samples at the same dilution level may be tested. 6.2.5.4.2. Negative results at any level may indicate a hook effect, and the allergen concentration where a hook effect is seen must be reported in the kit instructions. 6.2.5.4.3. If all dilution levels provide negative results, additional dilutions must be prepared and analyzed until the concentration at which the hook effect disappears (i.e., the upper 1012 The independent laboratory validation study, conducted under PTM program guidance, should verify 1013 the analytical results obtained in the method developer study in a controlled laboratory setting. 1014 6.3.2. Matrix Study 1015 An independent laboratory, approved by AOAC, will perform a matrix study, as described in 6.2.3, for at 1016 least one food matrix for every five matrices claimed. Food matrices do not include cleaning and 1017 sanitation solutions such as CIP rinse water. The independent laboratory must analyze at least one 1018 environmental surface/ cleaning and sanitizing solution for every five environmental surfaces/ cleaning 1019 and sanitizing solutions claims. The selection of which matrices/surfaces/ cleaning and sanitizing 1020 solutions are analyzed should be reflective of the range of difficulty associated with the claimed 1021 matrices. 1022 6.4. Collaborative Study Interlaboratory Validation 1023 concentration at which positive results are obtained) is determined. 6.3. Independent Laboratory Validation (PTM) 6.3.1. Scope