Allergens are bound on the surface of each well of the microtiter plate. For testing, a diluted sample (serum or plasma) is added to the wells. Specific IgE antibodies of the sample bind to the antigens in the wells during incubation, and non-bound components of serum or plasma are washed away after incubation. Subsequently, digoxigenin-labeled anti-human IgE antibodies and radish peroxidase coupled anti-digoxigenin antibodies (conjugate) are added.

The digoxigenin-labeled anti-human IgE antibodies bind to IgE antibodies of the sample, standards and controls. The conjugate binds to the digoxigenin-labeled anti-human IgE antibodies. Subsequently, un-bound detection antibodies are washed away. A color substrate TMB (3,3‘, 5,5‘-tetramethylbenzidine) is added, which is converted by the horseradish peroxidase conjugate bound enzyme. 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 dye formed is quantified photometrically by measuring the extinction. The quantification of the tests is carried out using a point-to-point regression of a 6-point standard curve.

Standards are calibrated according to the WHO reference serum 75/5021. The identified units/mL can be assigned to the respective CAP-classes and provide the level of specific IgE sensitization (See table below).