AOAC Gluten Quantitative Validation Guidance-Round 1(Nov 2023)

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ROBUSTNESS STUDIES

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Quantitative Assays 333 For the purpose of the examples provided below, we are using hypothetical assays with written 334 procedures that call for some or all of the following assay parameters, which will be referred to as 335 “factors” going forward: a 2g sample size, a 30-minute extraction, an extraction temperature of 60 o C, a 336 diluted sample extract that is loaded onto the ELISA plate in a 100 microliter volume, a 1:10 dilution of 337 the conjugated antibody in conjugate buffer, and/or a 20 minute substrate (e.g. TMB) incubation. 338 Remember that each factor should be varied both up and down by at least 20%. 339 Robustness study designs can be large, and may need to be broken up across test kits, operators, days 340 or other experimental conditions. The factorial treatment combinations (the Runs comprising specific 341 combinations of parameters as shown in the tables below) should be assigned randomly to each set of 342 conditions (test kit, day, operator, etc.). 343 Full Factorial (2 y ) Designs 344 For assays with just a few factors to vary, a full factorial design (also called a 2 y design) may be used. 345 These designs allow for the analysis of the effects of changes to each individual factor, as well as the 346 analysis of any interactions between factors. Examples of designs where two, three or four factors are 347 varied are given below. The row labeled “N” at the bottom of each table describes the “normal” factor 348 values for the assay, but this is only included for informational purposes and does not need to be tested 349 as part of the experiment. 350 Varying 2 Parameters Varying 3 Parameters Varying 4 Parameters Run Sample Size Extract Time Run Sample Size Extract Time Extract Temp Run Sample Size Extract. Time Extract Temp Sample Load 1 1.5g 20 min 1 1.5g 20 min 45 C 1 1.5g 20 min 45 C 50 µ l 2 2.5g 20 min 2 2.5g 20 min 45 C 2 2.5g 20 min 45 C 50 µ l 3 1.5g 40 min 3 1.5g 40 min 45 C 3 1.5g 40 min 45 C 50 µ l 4 2.5g 40 min 4 2.5g 40 min 45 C 4 2.5g 40 min 45 C 50 µ l N 2g 30 min 5 1.5g 20 min 75 C 5 1.5g 20 min 75 C 50 µ l Table 1. 6 2.5g 20 min 75 C 6 2.5g 20 min 75 C 50 µ l 7 1.5g 40 min 75 C 7 1.5g 40 min 75 C 50 µ l 8 2.5g 40 min 75 C 8 2.5g 40 min 75 C 50 µ l N 2g 30 min 60 C 9 1.5g 20 min 45 C 150 µ l Table 2. 10 2.5g 20 min 45 C 150 µ l 11 150 µ l 40 min 45 C 150 µ l 12 150 µ l 40 min 45 C 150 µ l 13 150 µ l 20 min 75 C 150 µ l 14 150 µ l 20 min 75 C 150 µ l 15 150 µ l 40 min 75 C 150 µ l 16 150 µ l 40 min 75 C 150 µ l N 2g 30 min 60 C 100 µ l Table 3.

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Fractional Factorial Designs