AOAC Guidance on FA Immunoassay Validation (August 2023)

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6.2.2.1.3. Method developers must make a good-faith effort to obtain the relevant foods listed in Tables 2-4. If the method developer feels they are unable to secure any particular food type, they should detail efforts to procure the food in the study report. 6.2.2.1.4. Method developers are advised to take extreme care in selecting, sorting, and preparing samples for the cross-reactivity testing to avoid analyzing samples with existing undeclared target food allergen residues. For example, it may be advisable to obtain whole nuts, seeds, spices, etc. to facilitate hand sorting, washing/shelling,

and grinding/milling.

6.2.2.1.5. To identify possible allergen cross-contact, it is advisable to screen commodities with

an alternative method (ELISA, PCR or other).

6.2.2.2. Cross-Reactivity

6.2.2.2.1. Study Design

6.2.2.2.1.1. One test portion of each blank food material in the cross-reactivity panel should be analyzed according to the entire method protocol to evaluate cross-

reactivity.

6.2.2.2.1.2. Cross-reactivity testing should be based on the full-strength extracts, i.e., a sample of the item being tested for cross-reactivity should be extracted using the extraction buffer and procedure outlined in the method instructions, then analyzed at full strength to determine if it leads to a positive result. (9) 6.2.2.2.1.3. Certain concentrated food ingredients may require dilution (e.g., colors, spices, gums, etc.). Such ingredients may be tested at a 10% concentration in a matrix such as rice flour. If this type of dilution is conducted during method validation, corresponding procedures for specific food ingredients must be stipulated in the method protocol, kit insert, validation certificate, and

validation report.

6.2.2.2.1.4. In general, food items tested for cross-reactivity should be prepared as they

would normally be analyzed.

6.2.2.2.1.5. Blank samples with a positive result should first be repeated with a second lot of the cross-reactive sample, and if the result persists it may also be evaluated with an alternative method (PCR, Western blot, mass spectrometry, alternate ELISA, etc.) to verify if the signal is the result of cross-reactivity or a true

positive due to cross-contact.

6.2.2.2.2. Data Analysis and Reporting

6.2.2.2.2.1. All results must be reported. If any analysis is repeated, both datasets must be

reported and a justification given for all repeat analysis.

6.2.2.2.3. Acceptance Criteria

6.2.2.2.3.1. Results of blank sample analysis are acceptable and not indicative of cross- reactivity if all blank samples in the cross-reactivity panel deliver results of

negative (not detected).

6.2.2.2.3.2. Cross-reactivity reporting: If cross-reactivity is observed, corresponding information must be included in the applicability statement of the kit insert,

validation report, and validation certificate.