Lars Yman, Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden

 

Pharmacia CAP System utilises a substandard to the WHO IgE standard as calibrator for serum IgE and for allergen-specific IgE antibodies.

 

The IgE molecule has a large number of epitopes on the Fc part. Combinations of catching-anti-IgE and conjugate-anti-IgE can be found, that makes the calibrator IgE and allergen-bound IgE appear identical. To what extent this applies to a certain assay can only be judged on the basis of experimental data and not on general assumptions. In case of Pharmacia CAP System it has been shown in model experiments with timothy pollen that the measurement of allergen-bound IgE is equivalent to IgE protein. Until it is convincingly proven that this is true for all allergens the specific antibody should be expressed in allergen specific units (UA). However, already the data summarized in Fig 1 clearly show that the IgE calibrator can be used for total as well as specific IgE and that the WHO unit can be a relevant unit allowing evaluation of IgE antibody concentration in mass units.

 

 

Fig. 1. IgE antibody and IgE protein in two sera after depletion of timothy specific antibody in four steps by timothy ImmunoCAP.

 

This is possible because of the fact that practically all the IgE antibodies of the sample are bound to the solid phase. It has been clearly demonstrated that in case of the timothy ImmunoCAP the capacity to bind IgE antibodies is 25 times bigger than what is required to bind all IgE antibodies at a concentration corresponding to the high end of the measuring range (100 kUA/l). This is a large excess and when applying the Law of Mass Action it is clear that formation of allergen-IgE complex is favored. The more allergen added - the more of the antibodies are bound to the allergen. In fact, since about 90 % or more of the IgE antibody of the sample is bound the excess is large enough to make the antibody binding independent of the association constant of the interaction (the affinity), regardless of the IgE antibody concentration.

 

 

Fig. 2. Quantity of IgE antibody specifically retained from 50 µl serum by one ImmunoCAP.

 

Furthermore, as long as all the allergenic components are present on the solid phase and shown to be capable of binding IgE antibodies, the system can be accepted as a quantitative assay. (Fig 3).

 

Fig. 3. Immunoblotting of 3 sera with timothy specific IgE Ab before (A) and after (B) contact with a Timothy ImmunoCAP.

 

According to the rules of WHO for establishment of standards for biological materials a standard for IgE was established in 1968. The standard was assigned arbitrary mass units (International Units) and submitted to collaborative trials. Later the International Unit was shown to correspond to 2.42 nanograms pure IgE by parallel chemical and immunological measurement (Bazaral M and Hamburger RN: Standardization and stability of immunoglobulin E (IgE). J Allergy Clin Immunol 49:189-191 (1972)). The International Unit is well established for total IgE measurements all over the world. It is evident that measurements of specific IgE antibodies also should move towards increasing standardization and that the antibody concentrations ultimately should be expressed in the same mass unit of IgE protein.