AOAC Guidance on FA Immunoassay Validation (August 2023)

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1.5.10.5.

For instances where the standard method protocol only requires one ELISA well to be measured for each test portion, the intermediate precision standard deviation is equivalent to the square root of the total variance component, reported as total

SD in the ANOVA table.

1.5.10.5.1.

For the example dataset when analyst is nested within lot and test

= .

portion is nested within analyst:

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2. Limit of Detection and Limit of Quantification Estimation

Limits of detection and quantification should be estimated using methods that account for the relationship between concentration and variance commonly observed with immunoassays (i.e., where variance increases with concentration), as described in IUPAC recommendations (Currie

1999)

2.1. Limit of Detection (LOD)

2.1.1. Data Required: mean observed concentration and intermediate precision standard deviation from analysis of at least four analyte levels of each claimed matrix, including

blank/zero

2.1.2. Calculations:

2.1.2.1.

Calculate mean concentration and intermediate precision standard deviation (S i )

for each test material.