Microarray Technology Applied to Human Allergic Disease

Abstract

IgE antibodies serve as the gatekeeper for the release of mediators from sensitized (IgE positive) mast cells and basophils following a relevant allergen exposure which can lead to an immediate-type hypersensitivity (allergic) reaction. Purified recombinant and native allergens were combined in the 1990s with state of the art chip technology to establish the first microarray-based IgE antibody assay. Triplicate spots to over 100 allergenic molecules are immobilized on an amine-activated glass slide to form a single panel multi-allergosorbent assay. Human antibodies, typically of the IgE and IgG isotypes, specific for one or many allergens bind to their respective allergen(s) on the chip. Following removal of unbound serum proteins, bound IgE antibody is detected with a fluorophore-labeled anti-human IgE reagent. The fluorescent profile from the completed slide provides a sensitization profile of an allergic patient which can identify IgE antibodies that bind to structurally similar (cross-reactive) allergen families versus molecules that are unique to a single allergen specificity. Despite its ability to rapidly analyze many IgE antibody specificities in a single simple assay format, the chip-based microarray remains less analytically sensitive and quantitative than its singleplex assay counterpart (ImmunoCAP, Immulite). Microgram per mL quantities of allergen-specific IgG antibody can also complete with nanogram per mL quantities of specific IgE for limited allergen binding sites on the chip. Microarray assays, while not used in clinical immunology laboratories for routine patient IgE antibody testing, will remain an excellent research tool for defining sensitization profiles of populations in epidemiological studies.

Keywords: ISAC; IgE; allergen extract; component resolved diagnosis; human; immunoenzymetric assay; immunosorbent allergen chip; microarray; molecular allergen; serodiagnosis.

Figure 1

Four assay formats which employ recombinant (with and without carbohydrate cross-reactive determinants or CCDs) and native purified allergens. CCDs confound specificity assessment especially with Hymenoptera venom specific IgE assays. Thus, the use of allergenic molecules without CCDs aid in more accurately defining the true allergenic specificity of the IgE antibody response. The microarray multiplex format (left assay) is used by the immunosorbent allergen chip or ISAC. It involves 112 individual allergens in triplicate arrays that are immobilized on an amine-activated chip. * A mixture of components from a single allergen specificity can be used to mimic an unpurified extract that often has extraneous non-allergenic material together with allergens. The arrows indicate the type of allergosorbents where recombinant and natural allergenic molecules can be effectively used. Adapted with permission from [8].