AOAC GFA Stakeholder Program Meeting Book (March 15, 2023)

4.1.7 High-Dose Hook Effect Study To determine whether the method is adversely affected by high concentrations of the analyte, a “hook effect” study must be performed. For each gluten source claimed, perform an extraction of a whole flour (or higher concentrate if commercially available, such as vital wheat gluten) per the kit instructions. Dilute this extract 1:10, 1:100, 1:1000 and 1:10,000 in the kit extraction buffer. Follow the kit instructions to prepare these extracts for testing, and then test each sample. Repeat this process four more times, resulting in five separate biological replicates for the whole gluten source and each dilution. All results are expected to be positive (or potentially invalid, for methods that have a ‘hook line’ indicating an excessive amount of analyte). In the event of any negative result, additional samples may be tested. Negative results at any level may indicate a hook effect, and any gluten concentration where a hook effect is seen must be reported in the kit instructions.

Page 7 – hook effect – terms “biological replicate” should be “test portions”