Competitive Inhibition
Methods in Allergy Diagnostics

Atopic allergy arises from an immune response mediated by IgE antibodies specific for substances called allergens. The diversity of individual IgE responses combined with the natural biological variability of allergen extracts complicates the diagnosis of atopic allergy. Methods that characterize the diversity of both extracts and IgE responses facilitate allergy diagnosis and help to evaluate and standardize allergy diagnostic assays. To assess the variability of allergen extracts, standard biochemistry techniques describe the extracts in terms of their total protein and carbohydrate content. To evaluate the IgE response to an allergen extract, the method of in vitro allergen inhibition describes the IgE-binding specificity of an extract.

The diagnosis of allergy requires evidence of specific IgE antibodies. Reactivity to a given allergenic substance, such as ragweed pollen, varies greatly among patients in the strength and diversity of the antibody response. While researchers have identified and described many important allergenic molecules, most reactive substances in allergen extracts remain uncharacterized. Typical extract diversity and the variability in patient responses prohibit complete characterization of allergen extracts and call for tools to evaluate extract composition and patient immune responses.

Competitive allergen inhibition assays are such tools. The FDA and immunotherapy extract manufacturers first adopted allergen inhibition methods to assess bioactivity of allergen extracts. Patients can also benefit from inhibition techniques: inhibition methods can evaluate potential immunotherapy reagents against individual patient serum, assuring high specificity in the immunotherapy extracts. In situations where the primary sensitizing allergen may be in question, inhibition using cross-reactive allergens may help identify it.

Competitive inhibition assays compare two allergen extracts by assessing the ability of one to block or reduce IgE binding to the other. The degree to which the extract inhibits IgE binding to the reference materials indicates the similarity between the two materials. Immunoblots, like inhibition assays, rely on IgE binding and reflect specific IgE reactivity. Extract components are separated by gel electrophoresis, then transferred to nitrocellulose, where IgE binds to the protein bands in the nitrocellulose. The pattern illustrates the number and scope of IgE specificities in the serum. (See Figure 1.) Immunoblots show complex reactivity patterns and reflect diversity in extract preparations. Practical applications of competitive inhibition include allergen quality control, confirmation testing, allergen content estimation, and identification of allergen cross-reactivity.

This technical report is also available as an online document in the Documents section.

Figure 1. DPC AlaBLOT®* specific IgE inhibition. Increasing amounts of inhibitor extract (timothy grass, G6) block IgE binding to the nitrocellulose AlaBLOT strips. The strips were incubated with a Class 3 positive serum pool.

*Available outside the US. In the US, sold as Analyte Specific Reagents.