AOAC Guidance on FA Immunoassay Validation (August 2023)

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variance from day/operator as one confounded variable, and variance from ELISA alone (i.e., well-to-well variance). In order for the nested designs to be capable of estimating repeatability, at least two test portions must be analyzed under repeatability conditions (i.e., conducted on the same day, by the same operator, with the same calibration and equipment). Under these conditions, the nested designs can estimate both intermediate precision and repeatability because repeatability is a variance component within intermediate precision, as expressed in the following equation, where s i 2 is the

intermediate precision variance, s lot

2 is the variance contributed by test kit lot,

s d/op 2 is the variance from the confounded factor of day and operator, and s r 2

is the repeatability variance:

2 = 2 + / 2 + 2

5.2.4.2.3.2. Repeatability estimates are required at four concentrations for each claimed matrix: blank, low, medium, and high levels, according to the claimed method

quantification range.

5.2.4.2.3.3. As intermediate precision estimates are used for the calculation of LOD and LOQ, estimates are required for all matrices, with at least three concentration

levels per matrix: blank, low, and medium.

5.2.4.2.3.4. Test kit lot variance (product consistency and stability) must be evaluated for at least one matrix using three test kit lots. This can be included in the estimation of intermediate precision (Designs 1b and 2b) or may be conducted separately (see Robustness Study, 5.2.5). Guidance on selection of test kit lots for product consistency and stability analysis can be found in

section 5.2.5.2.2.

5.2.4.2.3.5. Design 1a

5.2.4.2.3.5.1. Design 1a ( Figure 1 ) can be used to estimate (1) intermediate precision (which includes repeatability, test kit lot variance (with 1 degree of

freedom, df), and day/operator confounded variance) and (2)

repeatability.

5.2.4.2.3.5.2. 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 the test material, with one ELISA measurement performed per

test portion.