Allergen extracts from different foods are bound to the surface of the microtiter plate wells. For testing, a diluted sample (serum or plasma) is added to the wells. Specific IgG₄ antibodies of the sample bind to the antigens in the wells during incubation. Non-bound components of serum or plasma are washed away after incubation of the sample. Horseradish peroxidase- coupled anti-IgG₄ antibodies (conjugate) are added and bind to the IgG₄ antibodies of the sample, the standards or controls. Subsequently, unbound detection antibodies are washed away. The color substrate TMB (3,3‘,5,5‘-tetramethylbenzidine) is added, which is converted by the horseradish peroxidase conjugate. The reaction is stopped by adding a stop solution. A yellow dye is formed and the respective intensity correlates with the proportional amount of locally-bound antibody.

The identified units/mL can be assigned to the respective intensity and provide the level of specific IgG₄ sensitization (See table below).