AOAC Guidance on FA Immunoassay Validation (August 2023)

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5.2.3.2.1.1. One test portion of each blank food material in the selectivity panel should be analyzed according to the entire method protocol to evaluate cross-reactivity. 5.2.3.2.1.2. 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) 5.2.3.2.1.3. 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.

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

would normally be analyzed (raw or cooked).

5.2.3.2.1.5. If a positive result is obtained or it is outside the measurement range of the method, the extract must be diluted and rerun to characterize the extent of

the cross-reactivity.

5.2.3.2.1.6. Blank samples with a positive result (> LOD/LOQ) 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 whether the signal is the result of

cross-reactivity or a true positive due to cross-contact.

5.2.3.2.2. Data Analysis and Reporting

5.2.3.2.2.1. The absorbance or optical density (OD) values for all blank samples must be reported. The absorbance values for the following method standards analyzed with the blank samples also must be given: zero standard, first non-zero 5.2.3.2.2.2. The concentration for all blank samples that had an absorbance or OD above the limit of quantification of the method must be reported. Results falling between the LOD and LOQ must be reported as such. All results must be reported. If any analysis is repeated, both datasets must be reported, and a standard.

justification given for all repeat analysis.

5.2.3.2.3. Acceptance Criteria

5.2.3.2.3.1. Results of blank sample analysis are acceptable and not indicative of cross- reactivity if the extrapolated quantitative result of the blank sample is < LOQ.

5.2.3.2.3.2. Cross-reactivity reporting

5.2.3.2.3.2.1. If cross-reactivity is observed, corresponding information must be included in the applicability statement of the kit insert, validation report,

and validation certificate.

5.2.3.2.3.2.2. If the extrapolated quantitative result is between the LOD and LOQ, this must be described in the validation report but does not need to be stated

as cross-reactivity in the kit insert.