AOAC Guidance on FA Immunoassay Validation (August 2023)

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5.2.4.2.3.7. Design 2a

5.2.4.2.3.7.1. Design 2a ( Figure 3 ) can be used to estimate (1) intermediate precision (which includes repeatability, test kit lot variance (with 1 df), day/operator confounded variance, and ELISA variance), (2) repeatability (which includes test portion and ELISA variance), and (3) ELISA variance. 5.2.4.2.3.7.2. In this instance the repeatability variance can be further split into test portion variance and ELISA variance as shown in the equation below,

where s r

2 is repeatability variance, s TP

2 is the variance attributed to test

2 = 2 + 2

portion, s ELISA

2 is the variance attributed to ELISA measurement variance:

5.2.4.2.3.7.3. Two test kit lots are used to analyze each test material. Two operators conduct analysis on two days for each test kit lot. For each day and lot, the assigned operator conducts extraction and analysis of three test portions of test material, with two ELISA measurements performed per

test portion.

507 Figure 3: Design 2a. Lot: test kit lot, TP: test portion, E: ELISA measurement. Design 2a can be used to 508 estimate intermediate precision, repeatability, and ELISA variance.

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5.2.4.2.3.8. Design 2b

5.2.4.2.3.8.1. Design 2b ( Figure 4 ) can be used to estimate (1) intermediate precision (which includes repeatability, test kit lot variance (with 2 df), day/operator confounded variance, and ELISA variance), repeatability (which includes test portion variance and ELISA variance), (3) ELISA 5.2.4.2.3.8.2. Three test kit lots are used to analyze each test material. Two operators conduct analysis on two days for each test kit lot. For each day and lot, the assigned operator conducts extraction and analysis of two test portions of test material, with two ELISA measurements performed per variance, and (4) lot-to-lot product consistency.

test portion.